Peptide-based oral care surface reagents for personal care

ABSTRACT

Peptides have been identified that bind to oral surfaces such as teeth and gums. Peptide based oral care reagents have been created by couple such peptides to oral care benefit agents such as whiteners.

This patent application is a divisional of U.S. patent application Ser.No. 11/971,975, filed Jan. 10, 2008, now granted as U.S. Pat. No.7,807,141, which is a continuation-in-part of U.S. patent applicationSer. No. 11/074,473, filed Mar. 8, 2005, now abandoned, which is acontinuation-in-part of U.S. patent application Ser. No. 10/935,642,filed Sep. 7, 2004, now granted as U.S. Pat. No. 7,220,405, which claimsthe benefit of U.S. Provisional Application 60/501,498, filed Sep. 8,2003, now expired.

The invention relates to the field of personal care products. Morespecifically, the invention relates to oral care binding peptides aspart of a reagent for the delivery of oral care benefit agents to oralcavity surfaces.

BACKGROUND OF THE INVENTION

Film-forming substances are widely used in compositions for skin andhair care as conditioning agents and moisturizers, and to protect theskin and hair against environmental and chemical damage. Thesesubstances adsorb onto and/or absorb into the skin or hair, forming aprotective coating. Commonly used film-forming substances includesynthetic polymers, such as silicones, polyvinylpyrrolidone, acrylicacid polymers, and polysaccharides, and proteins, such as collagen,keratin, elastin, casein, silk, and soy proteins. Many proteins areknown to be particularly effective film-forming agents. Because of theirlow solubility at the conditions used in skin and hair care products,proteins are commonly used in the form of peptides, formed by thehydrolysis of the proteins.

In hair care and hair coloring compositions, film-forming substances areused to form a protective film on the surface of the hair to protect itfrom damage due to grooming and styling, shampooing, and exposure toultraviolet light and the reactive chemicals commonly used in permanentwave agents, hair coloring products, bleaches, and hair straighteners,which denature the hair keratin protein. Moreover, these film-formingsubstances improve the elasticity of the hair. Film-forming substancesthat have been used in hair care products include proteins, such askeratin, collagen, soy, and silk proteins and hydrolysates thereof, andpolymeric materials, such as polyacrylates, long chain alkyl quaternizedamines, and siloxane polymers. For example, Cannell et al. in U.S. Pat.No. 6,013,250 describe a hair care composition for treating hair againstchemical and ultraviolet light damage. That composition compriseshydrolyzed protein, having an abundance of anionic amino acids,particularly, sulfur-containing amino acids, and divalent cations. It isproposed in that disclosure that the anionic components of thehydrolyzed protein bind to the hair by means of cationic bridges. Aminoacids and their derivatives have also been used in hair carecompositions to condition and strengthen hair. For example, O'Toole etal. in WO 0051556 describe hair care compositions containing four ormore amino acid compounds selected from histidine, lysine, methionine,tyrosine, tryptophan, and cysteine compounds.

Film-forming substances are also used in skin care compositions to forma protective film on the skin. These films can serve to lubricate andcoat the skin to passively impede the evaporation of moisture and smoothand soften the skin. Commonly used film-forming substances in skin carecompositions include hydrolyzed animal and vegetable proteins (Puchalskiet al., U.S. Pat. No. 4,416,873, El-Menshawy et al., U.S. Pat. No.4,482,537, and Kojima et al., JP 02311412) and silk proteins (Philippeet al., U.S. Pat. No. 6,280,747 and Fahnestock et al., U.S. patentapplication Ser. No. 10/704,337). Amino acids and derivatives have alsobeen used in skin care compositions as conditioning agents. For example,Kojima et al. in JP 06065049 describe skin care compositions containingamino acids and/or their derivatives and docosahexaenoic acid, its saltsor its esters.

Hair coloring agents may be divided into three categories, specifically,permanent, semi-permanent or direct, and temporary. The permanent hairdyes are generally oxidative dyes that provide hair color that lastsabout four to six weeks. These oxidative hair dyes consist of two parts,one part contains the oxidative dyes in addition to other ingredients,while the second part contains an oxidizing agent such as hydrogenperoxide. The two components are mixed immediately prior to use. Theoxidizing agent oxidizes the dye precursors, which then combine to formlarge color molecules within the hair shaft. Although the oxidative hairdyes provide long-lasting color, the oxidizing agents they contain causehair damage. The semi-permanent or direct hair dyes are preformed dyemolecules that are applied to the hair and provide color for about sixto twelve shampoos. This type of hair dye is gentler to the hair becauseit does not contain peroxides, but the hair color does not last as long.Some improved durability is achieved by the use of nanoparticle haircoloring materials with a particle size of 10 to 500 nm, as described byHensen et al. in WO 01045652. These nanoparticle hair coloring materialsare conventional direct hair dyes that are treated to obtain nanoscaledimensions and exhibit increased absorption into the hair. Temporaryhair dyes are coloring agents that are applied to the hair surface andare removed after one shampoo. It would be desirable to develop a haircoloring agent that provides the durability of the permanent hair dyeswithout the use of oxidizing agents that damage hair.

The major problem with the current skin care and hair care compositions,non-oxidative hair dyes, as well as nail coloring agents is that theylack the required durability required for long-lasting effects. For thisreason, there have been attempts to enhance the binding of the cosmeticagent to the hair, skin or nails. For example, Richardson et al. in U.S.Pat. No. 5,490,980 and Green et al. in U.S. Pat. No. 6,267,957 describethe covalent attachment of cosmetic agents, such as skin conditioners,hair conditioners, coloring agents, sunscreens, and perfumes, to hair,skin, and nails using the enzyme transglutaminase. This enzymecrosslinks an amine moiety on the cosmetic agent to the glutamineresidues in skin, hair, and nails. Similarly, Green et al. in WO 0107009describe the use of the enzyme lysine oxidase to covalently attachcosmetic agents to hair, skin, and nails.

In another approach, cosmetic agents have been covalently attached toproteins or protein hydrolysates. For example, Lang et al. in U.S. Pat.No. 5,192,332 describe temporary coloring compositions that contain ananimal or vegetable protein, or hydrolysate thereof, which containresidues of dye molecules grafted onto the protein chain. In thosecompositions, the protein serves as a conditioning agent and does notenhance the binding of the cosmetic agent to hair, skin, or nails.Horikoshi et al. in JP 08104614 and Igarashi et al. in U.S. Pat. No.5,597,386 describe hair coloring agents that consist of an anti-keratinantibody covalently attached to a dye or pigment. The antibody binds tothe hair, thereby enhancing the binding of the hair coloring agent tothe hair. Similarly, Kizawa et al. in JP 09003100 describe an antibodythat recognizes the surface layer of hair and its use to treat hair. Ahair coloring agent consisting of that anti-hair antibody coupled tocolored latex particles is also described. The use of antibodies toenhance the binding of dyes to the hair is effective in increasing thedurability of the hair coloring, but these antibodies are difficult andexpensive to produce. Terada et al. in JP 2002363026 describe the use ofconjugates consisting of single-chain antibodies, preferablyanti-keratin, coupled to dyes, ligands, and cosmetic agents for skin andhair care compositions. The single-chain antibodies may be preparedusing genetic engineering techniques, but are still difficult andexpensive to prepare because of their large size. Findlay in WO 00048558describes the use of calcin proteins, such as β-lactoglobulin, whichcontain a binding domain for a cosmetic agent and another binding domainthat binds to at least a part of the surface of a hair fiber or skinsurface, for conditioners, dyes, and perfumes. Again these proteins arelarge and difficult and expensive to produce.

Linter in U.S. Pat. No. 6,620,419 describes peptides grafted to a fattyacid chain and their use in cosmetic and dermopharmaceuticalapplications. The peptides described in that disclosure are chosenbecause they stimulate the synthesis of collagen; they are not specificbinding peptides that enhance the durability of hair and skinconditioners, and hair, nail, and skin colorants.

Since its introduction in 1985, phage display has been widely used todiscover a variety of ligands including peptides, proteins and smallmolecules for drug targets (Dixit, J. of Sci. & Ind. Research,57:173-183 (1998)). The applications have expanded to other areas suchas studying protein folding, novel catalytic activities, DNA-bindingproteins with novel specificities, and novel peptide-based biomaterialscaffolds for tissue engineering (Hoess, Chem. Rev. 101:3205-3218 (2001)and Holmes, Trends Biotechnol. 20:16-21 (2002)). Whaley et al. (Nature405:665-668 (2000)) disclose the use of phage display screening toidentify peptide sequences that can bind specifically to differentcrystallographic forms of inorganic semiconductor substrates.

A modified screening method that comprises contacting a peptide librarywith an anti-target to remove peptides that bind to the anti-target,then contacting the non-binding peptides with the target has beendescribed (Estell et al. WO 0179479, Murray et al. U.S. PatentApplication Publication No. 2002/0098524, and Janssen et al. U.S. PatentApplication Publication No. 2003/0152976). Using that method, a peptidesequence that binds to hair and not to skin, given as SEQ ID NO:1, and apeptide sequence that binds to skin and not hair, given as SEQ ID NO:2,were identified. Using the same method, Janssen et al. (WO 04048399)identified other skin-binding and hair-binding peptides, as well asseveral binding motifs. Although the potential use of these peptides inpersonal care applications is suggested in those disclosures, thecoupling of these peptides to coloring agents and conditioning agents toprepare high-affinity hair conditioners, skin conditioners, haircolorants, nail colorants and skin colorants is not described. A methodfor identifying high-affinity phage-peptide clones is also described inthose disclosures. The method involves using PCR to identify peptidesthat remain bound to the target after acid elution.

Reisch (Chem. Eng. News 80:16-21 (2002)) reports that a family ofpeptides designed to target an ingredient of specific human tissue hasbeen developed for personal care applications. However, no descriptionof peptide-based conditioners or coloring agents are disclosed in thatpublication.

One of the peptide binding sequences, given as SEQ ID NO:3, has beenreported for several other purposes. For example, Hupp et al. in WO02065134 disclose the peptide sequence SEQ ID NO:3 as a peptide for usein modulating the binding of a p53 polypeptide to a p300 polypeptide,useful for regulating the mammalian cell cycle or to induce or preventcell death. Liu et al. in U.S. Pat. No. 6,344,443 describe the use ofthat same peptide sequence to inhibit binding of tumor necrosis factoralpha to its receptor for preventing or reversing inflammatory changesin patients with arthritis and other inflammatory diseases. Anotherpeptide binding sequence, given as SEQ ID NO:4, was reported by Jagotaet al. in WO 03102020 as a carbon nanotube-binding peptide.

In view of the above, a need exists for body surface reagents andpersonal care products that provide improved durability for long lastingeffects and are easy and inexpensive to prepare.

Applicants have met the stated needs by identifying peptide sequencesusing phage display screening that specifically bind to body surfaces,such as, hair, skin, nails, teeth, gums, corneal tissue, and oral cavitysurfaces, with high affinity and using them to design peptide-based bodysurface reagents, such as, hair conditioners, skin conditioners, haircolorants, nail colorants, skin colorants, and oral care reagents.

SUMMARY OF THE INVENTION

The invention provides peptide sequences that bind with high affinity tooral cavity surfaces such as teeth and gums.

Accordingly, the invention provides tooth pellicle-binding peptides asset forth in SEQ ID NO: 106-125 and tooth enamel-biding peptidesselected from the group consisting of SEQ ID NO:126-145.

In another embodiment the invention provides a diblock, peptide-basedoral care reagent having the general structure (OBP)_(n)-OBA, wherein

a) OBP is an oral cavity surface-binding peptide;

b) OBA is an oral care benefit agent; and

c) n ranges from 1 to about 10,000.

In another embodiment of the invention provides a triblock,peptide-based oral care reagent having the general structure[(OBP)_(m)-S]_(n)-OBA, wherein

a) OBP is an oral cavity surface-binding peptide;

b) OBA is an oral care benefit agent;

c) S is a spacer;

d) m ranges from 1 to about 50; and

e) n ranges from 1 to about 10,000.

In another embodiment the invention provides a method for applying anoral care benefit agent to an oral surface comprising contacting theoral surface with the peptide based oral care reagent of either of theinvention, comprising an oral surface binding peptide and an oral carebenefit agent, with an oral surface under conditions whereby the oralcare reagent binding peptide adheres to the oral surface. In analternate embodiment the invention provides a method for applying anoral care benefit reagent to an oral cavity surface comprising the stepsof:

-   -   a) providing an oral care reagent selected from the group        consisting of:        -   i) (OBP)_(n)-OBA; and        -   ii) [(OBP)_(m)-S]_(k)-OBA wherein            -   1) OBP is an oral cavity surface-binding peptide;            -   2) OBA is an oral care benefit agent;            -   3) n ranges from 1 to about 10,000;            -   4) S is a spacer;            -   5) m ranges from 1 to about 50; and            -   6) k ranges from 1 to about 10,000;                -   and wherein the oral cavity surface-binding peptide                    is selected by a method comprising the steps of:        -   A) providing a library of combinatorial generated            phage-peptides;        -   B) contacting the library of (A) with a oral cavity surface            sample to form a reaction solution comprising;        -   (i) phage-peptide-oral cavity surface sample complex;        -   (ii) unbound oral cavity surface sample, and        -   (iii) uncomplexed peptides;        -   C) isolating the phage-peptide-oral cavity surface sample            complex of (B);        -   D) eluting the weakly bound peptides from the isolated            peptide complex of (C);        -   E) identifying the remaining bound phage-peptides either by            using the polymerase chain reaction directly with the            phage-peptide-oral cavity surface sample complex remaining            after step (D), or by infecting bacterial host cells            directly with the phage-peptide-oral cavity surface sample            complex remaining after step (D), growing the infected cells            in a suitable growth medium, and isolating and identifying            the phage-peptides from the grown cells, wherein the            phage-peptides are from about 7 to about 25 amino acids and            have a binding affinity for oral cavity surface sample,            measured as MB₅₀, equal to or less than 10⁻⁵ M; and    -   b) applying the oral care benefit agent of (a) to an oral cavity        surface for a time sufficient for the peptide-based oral care        agent to bind to an oral cavity surface.

BRIEF DESCRIPTION OF SEQUENCE DESCRIPTIONS

The invention can be more fully understood from the following detaileddescription and the accompanying sequence descriptions, which form apart of this application.

The following sequences conform with 37 C.F.R. 1.821-1.825(“Requirements for Patent Applications Containing Nucleotide Sequencesand/or Amino Acid Sequence Disclosures—the Sequence Rules”) andconsistent with World Intellectual Property Organization (WIPO) StandardST.25 (1998) and the sequence listing requirements of the EPO and PCT(Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of theAdministrative Instructions). The symbols and format used for nucleotideand amino acid sequence data comply with the rules set forth in 37C.F.R. §1.822.

SEQ ID NO:1 is the amino acid sequence of a hair-binding peptide.

SEQ ID NO:2 is the amino acid sequence of a skin-binding peptide.

SEQ ID NOs:3-52, 54-59 are the amino acid sequences of hair-bindingpeptides.

SEQ ID NO:53 is the amino acid sequence of a hair-binding andnail-binding peptide.

SEQ ID NO:60 is the amino acid sequence of a nail-binding peptide.

SEQ ID NO:61 is the amino acid sequence of a skin-binding peptide.

SEQ ID NO:62 is the oligonucleotide primer used to sequence phage DNA.

SEQ ID NO:63 is the amino acid sequence of a peptide used as a controlin the ELISA binding assay.

SEQ ID NO:64 is the amino acid sequence of a cysteine-attachedhair-binding peptide.

SEQ ID NO:65 is the amino acid sequence of the Caspase 3 cleavage site.

SEQ ID NOs:66, 69, and 70 are the amino acid sequence ofshampoo-resistant hair-binding peptides.

SEQ ID NOs:67 and 68 are the nucleotide sequences of the primers used toamplify shampoo-resistant, hair-binding phage peptides, as described inExample 8.

SEQ ID NOs:71-74 are the amino acid sequences of the biotinylatedhair-binding and skin-binding peptides used Example 9.

SEQ ID NO:75 is the amino acid sequence of the fully protected D21peptide used in Example 16.

SEQ ID NOs:76-98 are the amino acid sequences of hair-binding peptides.

SEQ ID NOs:99-104 are the amino acid sequences of skin-binding peptides.

SEQ ID NO:105 is a primer sequence.

SEQ ID NOs: 106-125 are tooth pellicle binding peptide amino acidsequences.

SEQ ID NOs: 126-145 are tooth enamel binding peptide amino acidsequences.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides peptide sequences that specifically bindto human body surfaces such as hair, skin, nails, teeth, gums, and thelike with high affinity. Additionally, the present invention providespeptide based body surface reagents that are comprised of body surfacebinding peptides coupled with various benefit agents that convey abenefit to the body surface. Typical of the compositions arepeptide-based hair and skin conditioners, and hair, nail, and skincolorants with improved durability.

The peptide based body surface reagents of the invention providebenefits and an advance over the art in the development of personal careproducts. Because the reagents are peptide based they are able to bindstrongly to surfaces from an aqueous environment, thus in many casesbeing both water soluble and water fast. Additionally, because of theaqueous nature of the reagents they may be removed from body surfaceswithout of the use of odor producing chemicals. The reagents of theinvention bind almost immediately to the target body surface,eliminating the need for long drying times, typical of most personalcare applications. Additionally the reagents of the invention arespecific in their affinity for body surfaces, making the need to isolatetheir application to a specific surface unnecessary. Thus a regent thatbinds hair for coloring will not bind skin and visa versa. Mostimportantly, the peptide nature of the reagents makes them virtuallynon-toxic and non-irritating to exposed body surfaces such as the skinand the membranes of the eyes and mouth.

The following definitions are used herein and should be referred to forinterpretation of the claims and the specification.

“HBP” means hair-binding peptide.

“SBP” means skin-binding peptide.

“NBP” means nail-binding peptide.

“OBP” means oral cavity surface-binding peptide.

“TBP” means tooth-binding peptide.

“HCA” means hair conditioning agent.

“SCA” means skin conditioning agent.

“C” means coloring agent for hair, skin, or nails.

“OBA” means oral benefit agent.

“S” means spacer.

“BSBP” means body surface binding peptide.

“BA” means benefit agent.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having,” “contains” or “containing,” or any othervariation thereof, are intended to cover a non-exclusive inclusion. Forexample, a composition, a mixture, process, method, article, orapparatus that comprises a list of elements is not necessarily limitedto only those elements but may include other elements not expresslylisted or inherent to such composition, mixture, process, method,article, or apparatus. Further, unless expressly stated to the contrary,“or” refers to an inclusive or and not to an exclusive or. For example,a condition A or B is satisfied by any one of the following: A is true(or present) and B is false (or not present), A is false (or notpresent) and B is true (or present), and both A and B are true (orpresent).

Also, the indefinite articles “a” and “an” preceding an element orcomponent of the invention are intended to be nonrestrictive regardingthe number of instances (i.e. occurrences) of the element or component.Therefore “a” or “an” should be read to include one or at least one, andthe singular word form of the element or component also includes theplural unless the number is obviously meant to be singular.

As used herein, the term “about” refers to modifying the quantity of aningredient or reactant of the invention or employed refers to variationin the numerical quantity that can occur, for example, through typicalmeasuring and liquid handling procedures used for making concentrates oruse solutions in the real world; through inadvertent error in theseprocedures; through differences in the manufacture, source, or purity ofthe ingredients employed to make the compositions or carry out themethods; and the like. The term “about” also encompasses amounts thatdiffer due to different equilibrium conditions for a compositionresulting from a particular initial mixture. Whether or not modified bythe term “about”, the claims include equivalents to the quantities.

The term “invention” or “present invention” as used herein is anon-limiting term and is not intended to refer to any single embodimentof the particular invention but encompasses all possible embodiments asdescribed in the specification and the claims.

The term “peptide” refers to two or more amino acids joined to eachother by peptide bonds or modified peptide bonds.

The term “body surface” will mean any surface of the human body that mayserve as a substrate for the binding of a peptide carrying a benefitagent. Typical body surfaces include but are not limited to hair, skin,nails, teeth, gums, and corneal tissue.

The term “benefit agent’ is a general term applying to a compound orsubstance that may be coupled with a binding peptide for application toa body surface. Benefit agents typically include conditioners,colorants, fragrances, whiteners and the like along with othersubstances commonly used in the personal care industry.

The term “hair” as used herein refers to human hair, eyebrows, andeyelashes.

The term “skin” as used herein refers to human skin, or pig skin,VITRO-SKIN® and EPIDERM™ which are substitutes for human skin. Skin asused herein as a body surface will generally comprise a layer ofepithelial cells and may additionally comprise a layer of endothelialcells.

The term “nails” as used herein refers to human fingernails andtoenails.

The terms “coupling” and “coupled” as used herein refer to any chemicalassociation and includes both covalent and non-covalent interactions.

The term “stringency” as it is applied to the selection of thehair-binding, skin-binding, oral cavity-binding, teeth-binding, andnail-binding peptides of the present invention, refers to theconcentration of the eluting agent (usually detergent) used to elutepeptides from the hair, skin, oral cavity surface, teeth or nails.Higher concentrations of the eluting agent provide more stringentconditions.

The term “peptide-body surface sample complex” means structurecomprising a peptide bound to a sample of a body surface via a bindingsite on the peptide.

The term “peptide-hair complex” means structure comprising a peptidebound to a hair fiber via a binding site on the peptide.

The term “peptide-skin complex” means structure comprising a peptidebound to the skin via a binding site on the peptide.

The term “peptide-nail complex” means structure comprising a peptidebound to fingernails or toenails via a binding site on the peptide.

The term “peptide-oral cavity surface complex” means structurecomprising a peptide bound to an oral cavity surface via a binding siteon the peptide.

The term “peptide-substrate complex” refers to either peptide-hair,peptide-skin, peptide-oral cavity surface or peptide-nail complexes.

The term “MB₅₀” refers to the concentration of the binding peptide thatgives a signal that is 50% of the maximum signal obtained in anELISA-based binding assay, as described in Example 9. The MB₅₀ providesan indication of the strength of the binding interaction or affinity ofthe components of the complex. The lower the value of MB₅₀, the strongerthe interaction of the peptide with its corresponding substrate.

The term “binding affinity” refers to the strength of the interaction ofa binding peptide with its respective substrate. The binding affinity isdefined herein in terms of the MB₅₀ value, determined in an ELISA-basedbinding assay.

The term “nanoparticles” are herein defined as particles with an averageparticle diameter of between 1 and 100 nm. Preferably, the averageparticle diameter of the particles is between about 1 and 40 nm. As usedherein, “particle size” and “particle diameter” have the same meaning.Nanoparticles include, but are not limited to, metallic, semiconductor,polymer, or silica particles.

The term “amino acid” refers to the basic chemical structural unit of aprotein or polypeptide. The following abbreviations are used herein toidentify specific amino acids:

Three-Letter One-Letter Amino Acid Abbreviation Abbreviation Alanine AlaA Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys CGlutamine Gln Q Glutamic acid Glu E Glycine Gly G Histidine His HIsoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met MPhenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences “Chimeric gene” refers to any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. A “foreign” gene refers to a gene not normally found in thehost organism, but that is introduced into the host organism by genetransfer. Foreign genes can comprise native genes inserted into anon-native organism, or chimeric genes.

“Synthetic genes” can be assembled from oligonucleotide building blocksthat are chemically synthesized using procedures known to those skilledin the art. These building blocks are ligated and annealed to form genesegments which are then enzymatically assembled to construct the entiregene. “Chemically synthesized”, as related to a sequence of DNA, meansthat the component nucleotides were assembled in vitro. Manual chemicalsynthesis of DNA may be accomplished using well-established procedures,or automated chemical synthesis can be performed using one of a numberof commercially available machines. Accordingly, the genes can betailored for optimal gene expression based on optimization of nucleotidesequence to reflect the codon bias of the host cell. The skilled artisanappreciates the likelihood of successful gene expression if codon usageis biased towards those codons favored by the host. Determination ofpreferred codons can be based on a survey of genes derived from the hostcell where sequence information is available.

“Coding sequence” refers to a DNA sequence that codes for a specificamino acid sequence. “Suitable regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, polyadenylationrecognition sequences, RNA processing site, effector binding site andstem-loop structure.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.Promoters which cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”. It isfurther recognized that since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofdifferent lengths may have identical promoter activity.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide.

The term “transformation” refers to the transfer of a nucleic acidfragment into the genome of a host organism, resulting in geneticallystable inheritance. Host organisms containing the transformed nucleicacid fragments are referred to as “transgenic” or “recombinant” or“transformed” organisms.

The term “host cell” refers to cell which has been transformed ortransfected, or is capable of transformation or transfection by anexogenous polynucleotide sequence.

The terms “plasmid”, “vector” and “cassette” refer to an extrachromosomal element often carrying genes which are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA molecules. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that facilitate transformation of a particular host cell.“Expression cassette” refers to a specific vector containing a foreigngene and having elements in addition to the foreign gene that allow forenhanced expression of that gene in a foreign host.

The term “phage” or “bacteriophage” refers to a virus that infectsbacteria. Altered forms may be used for the purpose of the presentinvention. The preferred bacteriophage is derived from the “wild” phage,called M13. The M13 system can grow inside a bacterium, so that it doesnot destroy the cell it infects but causes it to make new phagescontinuously. It is a single-stranded DNA phage.

The term “phage display” refers to the display of functional foreignpeptides or small proteins on the surface of bacteriophage or phagemidparticles. Genetically engineered phage may be used to present peptidesas segments of their native surface proteins. Peptide libraries may beproduced by populations of phage with different gene sequences.

“PCR” or “polymerase chain reaction” is a technique used for theamplification of specific DNA segments (U.S. Pat. Nos. 4,683,195 and4,800,159).

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described by Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989) (hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M. L.and Enquist, L. W., Experiments with Gene Fusions, Cold Spring HarborLaboratory Cold Press Spring Harbor, N.Y. (1984); and by Ausubel, F. M.et al., Current Protocols in Molecular Biology, published by GreenePublishing Assoc. and Wiley-Interscience (1987).

Body Surfaces

Body surfaces of the invention are any surface on the human body thatwill serve as a substrate for a binding peptide. Typical body surfacesinclude, but are not limited to hair, skin, nails, teeth, gums, cornealtissue and the tissues of the oral cavity. In many cases the bodysurfaces of the invention will be exposed to air, however in someinstances, the oral cavity for example, the surfaces will be internal.Accordingly body surfaces may include layers of both epithelial and wellas endothelial cells.

Samples of body surfaces are available from a variety of sources. Forexample, human hair samples are available commercially, for example fromInternational Hair Importers and Products (Bellerose, N.Y.), indifferent colors, such as brown, black, red, and blond, and in varioustypes, such as African-American, Caucasian, and Asian. Additionally, thehair samples may be treated for example using hydrogen peroxide toobtain bleached hair. Pig skin, available from butcher shops andsupermarkets, VITRO-SKIN®, available from IMS Inc. (Milford, Conn.), andEPIDERM™, available from MatTek Corp. (Ashland, Mass.), are goodsubstitutes for human skin. Human fingernails and toenails may beobtained from volunteers. Extracted human teeth and false teeth may beobtained from Dental offices. Hydroxyapatite (Berkeley AdvancedBiomaterials, Inc. (San Leandro, Calif.)) can be coated with salivaryglycoproteins to mimic a natural tooth pellicle surface (tooth enamel ispredominantly comprised of hydroxyapatite).

Body Surface-Binding Peptides

Body surface-binding peptides as defined herein are peptide sequencesthat specifically bind with high affinity to specific body surfaces,including, but not limited to hair, skin, nails, teeth, tongue, cheeks,lips, gums, corneal tissue and the tissues of the oral cavity, forexample. Body surface-binding peptides of the present invention are fromabout 7 amino acids to about 45 amino acids, more preferably, from about7 amino acids to about 20 amino acids. The binding peptides of theinvention have a binding affinity for their respective substrate, asmeasured by MB₅₀ values, of less than or equal to about 10⁻² M, lessthan or equal to about 10⁻³ M, less than or equal to about 10⁻⁴ M, lessthan or equal to about 10⁻⁵ M, preferably less than or equal to about10⁻⁶ M, and more preferably less than or equal to about 10⁻⁷ M.

Suitable body surface-binding peptide sequences may be selected usingmethods that are well known in the art. The peptides of the presentinvention are generated randomly and then selected against a specificbody surface, for example, hair, skin, nail, or oral cavity surfacesample, based upon their binding affinity for the surface of interest.The generation of random libraries of peptides is well known and may beaccomplished by a variety of techniques including, bacterial display(Kemp, D. J.; Proc. Natl. Acad. Sci. USA 78(7):4520-4524 (1981), andHelfman et al., Proc. Natl. Acad. Sci. USA 80(1):31-35, (1983)), yeastdisplay (Chien et al., Proc Natl Acad Sci USA 88(21):9578-82 (1991)),combinatorial solid phase peptide synthesis (U.S. Pat. No. 5,449,754,U.S. Pat. No. 5,480,971, U.S. Pat. No. 5,585,275, U.S. Pat. No.5,639,603), and phage display technology (U.S. Pat. No. 5,223,409, U.S.Pat. No. 5,403,484, U.S. Pat. No. 5,571,698, U.S. Pat. No. 5,837,500).Techniques to generate such biological peptide libraries are describedin Dani, M., J. of Receptor & Signal Transduction Res., 21(4):447-468(2001).

A preferred method to randomly generate peptides is by phage display.Phage display is an in vitro selection technique in which a peptide orprotein is genetically fused to a coat protein of a bacteriophage,resulting in display of fused peptide on the exterior of the phagevirion, while the DNA encoding the fusion resides within the virion.This physical linkage between the displayed peptide and the DNA encodingit allows screening of vast numbers of variants of peptides, each linkedto a corresponding DNA sequence, by a simple in vitro selectionprocedure called “biopanning”. In its simplest form, biopanning iscarried out by incubating the pool of phage-displayed variants with atarget of interest that has been immobilized on a plate or bead, washingaway unbound phage, and eluting specifically bound phage by disruptingthe binding interactions between the phage and the target. The elutedphage is then amplified in vivo and the process is repeated, resultingin a stepwise enrichment of the phage pool in favor of the tightestbinding sequences. After 3 or more rounds of selection/amplification,individual clones are characterized by DNA sequencing.

After a suitable library of peptides has been generated, they are thencontacted with an appropriate amount of the test substrate, specificallya body surface sample. The library of peptides is dissolved in asuitable solution for contacting the sample. The body surface sample maybe suspended in the solution or may be immobilized on a plate or bead. Apreferred solution is a buffered aqueous saline solution containing asurfactant. A suitable solution is Tris-buffered saline (TBS) with 0.5%TWEEN® 20. The solution may additionally be agitated by any means inorder to increase the mass transfer rate of the peptides to body surfacesample, thereby shortening the time required to attain maximum binding.

Upon contact, a number of the randomly generated peptides will bind tothe body surface sample to form a peptide-body-surface complex, forexample a peptide-hair, peptide-skin, peptide-nail, or peptide-oralcavity surface complex. Unbound peptide may be removed by washing. Afterall unbound material is removed, peptides having varying degrees ofbinding affinities for the test surface may be fractionated by selectedwashings in buffers having varying stringencies. Increasing thestringency of the buffer used increases the required strength of thebond between the peptide and body surface in the peptide-body surfacecomplex. A number of substances may be used to vary the stringency ofthe washing solution in the peptide selection process including, but notlimited to acids (pH 1.5-3.0), bases (pH 10-12.5), salts of highconcentrations such as MgCl₂ (3-5 M) and LiCl (5-10 M), ethylene glycol(25-50%), dioxane (5-20%), thiocyanate (1-5 M), guanidine (2-5 M), urea(2-8 M), and surfactants of various concentrations such as SDS (sodiumdodecyl sulfate), DOC (sodium deoxycholate), Nonidet P-40, Triton X-100,shampoo (useful when selecting peptides for use in personal carecompositions, such as a commercial shampoo formulation), TWEEN® 20,wherein TWEEN® 20 is more typical. These substances may be prepared inbuffer solutions including, but not limited to, Tris-HCl, Tris-bufferedsaline, Tris-borate, Tris-acetic acid, triethylamine, phosphate buffer,and glycine-HCl, wherein Tris-buffered saline solution is preferred. Itwill be appreciated that peptides having increasing binding affinitiesfor body surface substrates may be eluted by repeating the selectionprocess using buffers with increasing stringencies.

The eluted peptides can be identified and sequenced by any means knownin the art.

Thus, the following method for generating the body surface-bindingpeptides, for example, hair-binding peptides, skin-binding peptides,nail-binding peptides, or oral cavity surface-binding peptides, of thepresent invention was used. A library of combinatorial generatedphage-peptides is contacted with the body surface of interest, to formphage peptide-body surface complexes. The phage-peptide-body-surfacecomplex is separated from uncomplexed peptides and unbound substrate,and the bound phage-peptides from the phage-peptide-body surfacecomplexes is eluted from the complex, preferably by acid treatment.Then, the eluted peptides are identified and sequenced. To identifypeptide sequences that bind to one substrate but not to another, forexample peptides that bind to hair, but not to skin or peptides thatbind to skin, but not to hair, a subtractive panning step is added.Specifically, the library of combinatorial generated phage-peptides isfirst contacted with the non-target to remove phage-peptides that bindto it. Then, the non-binding phage-peptides are contacted with thedesired substrate and the above process is followed. Alternatively, thelibrary of combinatorial generated phage-peptides may be contacted withthe non-target and the desired substrate simultaneously. Then, thephage-peptide-body surface complexes are separated from thephage-peptide-non-target complexes and the method described above isfollowed for the desired phage-peptide-body surface complexes.

One embodiment of the present invention provides a modified phagedisplay screening method for isolating peptides with a higher affinityfor body surfaces. In the modified method, the phage-peptide-bodysurface complexes are formed as described above. Then, these complexesare treated with an elution buffer. Any of the elution buffers describedabove may be used. Preferably, the elution buffer is an acidic solution.Then, the remaining, elution-resistant phage-peptide-body surfacecomplexes are used to directly infect a bacterial host cell, such as E.coli ER2738. The infected host cells are grown in an appropriate growthmedium, such as LB (Luria-Bertani) medium, and this culture is spreadonto agar, containing a suitable growth medium, such as LB medium withIPTG (isopropyl β-D-thiogalactopyranoside) and S-GAL™. After growth, theplaques are picked for DNA isolation and sequencing to identify thepeptide sequences with a high binding affinity for the body surface ofinterest.

In another embodiment, PCR may be used to identify the elution-resistantphage-peptides from the modified phage display screening method,described above, by directly carrying out PCR on the phage-peptide-bodysurface complexes using the appropriate primers, as described by Janssenet al. in U.S. Patent Application Publication No. 2003/0152976, which isincorporated herein by reference.

Hair-binding, skin-binding, and nail-binding peptides have beenidentified using the above methods. Specifically, binding peptides wereisolated that have a high affinity for normal brown hair, given as SEQID NOs:3-18, 28-38, 40-56, and 64; shampoo resistant, normal brown hair,given as SEQ ID NO:66, 69 and 70; bleached hair, given as SEQ ID NOs:7,8, 19-27, 38-40, 43, 44, 47, 57, 58, and 59, fingernail, given as SEQ IDNOs:53 and 60; and skin, given as SEQ ID NO:61. Additionally, thefingernail-binding peptides were found to bind to bleached hair and maybe used in the peptide-based hair conditioners and hair colorants. Thebleached hair-binding peptides will bind to fingernails and may be usedin the peptide-based nail colorants.

Production of Binding Peptides

The binding peptides of the present invention may be prepared usingstandard peptide synthesis methods, which are well known in the art (seefor example Stewart et al., Solid Phase Peptide Synthesis, PierceChemical Co., Rockford, Ill., 1984; Bodanszky, Principles of PeptideSynthesis, Springer-Verlag, New York, 1984; and Pennington et al.,Peptide Synthesis Protocols, Humana Press, Totowa, N.J., 1994).Additionally, many companies offer custom peptide synthesis services.

Alternatively, the peptides of the present invention may be preparedusing recombinant DNA and molecular cloning techniques. Genes encodingthe hair-binding, skin-binding or nail-binding peptides may be producedin heterologous host cells, particularly in the cells of microbialhosts.

Preferred heterologous host cells for expression of the binding peptidesof the present invention are microbial hosts that can be found broadlywithin the fungal or bacterial families and which grow over a wide rangeof temperature, pH values, and solvent tolerances. Becausetranscription, translation, and the protein biosynthetic apparatus arethe same irrespective of the cellular feedstock, functional genes areexpressed irrespective of carbon feedstock used to generate cellularbiomass. Examples of host strains include, but are not limited to,fungal or yeast species such as Aspergillus, Trichoderma, Saccharomyces,Pichia, Candida, Hansenula, or bacterial species such as Salmonella,Bacillus, Acinetobacter, Rhodococcus, Streptomyces, Escherichia,Pseudomonas, Methylomonas, Methylobacter, Alcaligenes, Synechocystis,Anabaena, Thiobacillus, Methanobacterium and Klebsiella.

A variety of expression systems can be used to produce the peptides ofthe present invention. Such vectors include, but are not limited to,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, frominsertion elements, from yeast episomes, from viruses such asbaculoviruses, retroviruses and vectors derived from combinationsthereof such as those derived from plasmid and bacteriophage geneticelements, such as cosmids and phagemids. The expression systemconstructs may contain regulatory regions that regulate as well asengender expression. In general, any system or vector suitable tomaintain, propagate or express polynucleotide or polypeptide in a hostcell may be used for expression in this regard. Microbial expressionsystems and expression vectors contain regulatory sequences that directhigh level expression of foreign proteins relative to the growth of thehost cell. Regulatory sequences are well known to those skilled in theart and examples include, but are not limited to, those which cause theexpression of a gene to be turned on or off in response to a chemical orphysical stimulus, including the presence of regulatory elements in thevector, for example, enhancer sequences. Any of these could be used toconstruct chimeric genes for production of the any of the bindingpeptides of the present invention. These chimeric genes could then beintroduced into appropriate microorganisms via transformation to providehigh level expression of the peptides.

Vectors or cassettes useful for the transformation of suitable hostcells are well known in the art. Typically the vector or cassettecontains sequences directing transcription and translation of therelevant gene, one or more selectable markers, and sequences allowingautonomous replication or chromosomal integration. Suitable vectorscomprise a region 5′ of the gene, which harbors transcriptionalinitiation controls and a region 3′ of the DNA fragment which controlstranscriptional termination. It is most preferred when both controlregions are derived from genes homologous to the transformed host cell,although it is to be understood that such control regions need not bederived from the genes native to the specific species chosen as aproduction host. Selectable marker genes provide a phenotypic trait forselection of the transformed host cells such as tetracycline orampicillin resistance in E. coli.

Initiation control regions or promoters which are useful to driveexpression of the chimeric gene in the desired host cell are numerousand familiar to those skilled in the art. Virtually any promoter capableof driving the gene is suitable for producing the binding peptides ofthe present invention including, but not limited to: CYC1, HIS3, GAL1,GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI (usefulfor expression in Saccharomyces); AOX1 (useful for expression inPichia); and lac, ara, tet, trp, IP_(L), IP_(R), T7, tac, and trc(useful for expression in Escherichia coli) as well as the amy, apr, nprpromoters and various phage promoters useful for expression in Bacillus.

Termination control regions may also be derived from various genesnative to the preferred hosts. Optionally, a termination site may beunnecessary, however, it is most preferred if included.

The vector containing the appropriate DNA sequence as described supra,as well as an appropriate promoter or control sequence, may be employedto transform an appropriate host to permit the host to express thepeptide of the present invention. Cell-free translation systems can alsobe employed to produce such peptides using RNAs derived from the DNAconstructs of the present invention. Optionally it may be desired toproduce the instant gene product as a secretion product of thetransformed host. Secretion of desired proteins into the growth mediahas the advantages of simplified and less costly purificationprocedures. It is well known in the art that secretion signal sequencesare often useful in facilitating the active transport of expressibleproteins across cell membranes. The creation of a transformed hostcapable of secretion may be accomplished by the incorporation of a DNAsequence that codes for a secretion signal which is functional in theproduction host. Methods for choosing appropriate signal sequences arewell known in the art (see for example EP 546049 and WO 9324631). Thesecretion signal DNA or facilitator may be located between theexpression-controlling DNA and the instant gene or gene fragment, and inthe same reading frame with the latter.

Peptide-Based Hair Conditioners

The peptide-based hair conditioners are formed by coupling ahair-binding peptide (HBP) with a hair conditioning agent (HCA). Thehair-binding peptide part of the conditioner binds strongly to the hair,thus keeping the conditioning agent attached to the hair for a longlasting conditioning effect. The hair-binding peptides include, but arenot limited to, hair-binding peptides selected by the screening methodsdescribed above, including the hair-binding peptide sequences, given bySEQ ID NOs: 3-59, 64, 66, 69, and 70, most preferably the peptides givenby SEQ ID NO:46 and SEQ ID NO:66, which bind strongly to hair, but notto skin. Additionally, any known hair-binding peptide may be used,including but not limited to SEQ ID NO:1, and SEQ ID NOs:76-98,described by Janssen et al. in U.S. Patent Application Publication No.2003/0152976 and by Janssen et al. in WO 04048399, respectively, both ofwhich are incorporated herein by reference. For bleached hair, thefingernail-binding peptide, given as SEQ ID NO:60, may also be used.

Hair conditioning agents as herein defined are agents which improve theappearance, texture, and sheen of hair as well as increasing hair bodyor suppleness. Hair conditioning agents, include, but are not limitedto, styling aids, hair straightening aids, hair strengthening aids, andvolumizing agents, such as nanoparticles. In the peptide-based hairconditioners of the present invention, any known hair conditioning agentmay be used. Hair conditioning agents are well known in the art, see forexample Green et al. (WO 0107009), incorporated herein by reference, andare available commercially from various sources. Suitable examples ofhair conditioning agents include, but are not limited to, cationicpolymers, such as cationized guar gum, diallyl quaternary ammoniumsalt/acrylamide copolymers, quaternized polyvinylpyrrolidone andderivatives thereof, and various polyquaternium-compounds; cationicsurfactants, such as stearalkonium chloride, centrimonium chloride, andSapamin hydrochloride; fatty alcohols, such as behenyl alcohol; fattyamines, such as stearyl amine; waxes; esters; nonionic polymers, such aspolyvinylpyrrolidone, polyvinyl alcohol, and polyethylene glycol;silicones; siloxanes, such as decamethylcyclopentasiloxane; polymeremulsions, such as amodimethicone; and nanoparticles, such as silicananoparticles and polymer nanoparticles. The preferred hair conditioningagents contain amine or hydroxyl functional groups to facilitatecoupling to the hair-binding peptides, as described below. Examples ofpreferred conditioning agents are octylamine (CAS No. 111-86-4), stearylamine (CAS No. 124-30-1), behenyl alcohol (CAS No. 661-19-8, CognisCorp., Cincinnati, Ohio), vinyl group terminated siloxanes, vinyl groupterminated silicone (CAS No. 68083-19-2), vinyl group terminated methylvinyl siloxanes, vinyl group terminated methyl vinyl silicone (CAS No.68951-99-5), hydroxyl terminated siloxanes, hydroxyl terminated silicone(CAS No. 80801-30-5), amino-modified silicone derivatives,[(aminoethyl)amino]propyl hydroxyl dimethyl siloxanes,[(aminoethyl)amino]propyl hydroxyl dimethyl silicones, andalpha-tridecyl-omega-hydroxy-poly(oxy-1,2-ethanediyl) (CAS No.24938-91-8).

The peptide-based hair conditioners are prepared by coupling a specifichair-binding peptide to a hair conditioning agent, either directly orvia an optional spacer. The coupling interaction may be a covalent bondor a non-covalent interaction, such as hydrogen bonding, electrostaticinteraction, hydrophobic interaction, or Van der Waals interaction. Inthe case of a non-covalent interaction, the peptide-based hairconditioner may be prepared by mixing the peptide with the conditioningagent and the optional spacer (if used) and allowing sufficient time forthe interaction to occur. The unbound materials may be separated fromthe resulting peptide-based hair conditioner adduct using methods knownin the art, for example, gel permeation chromatography.

The peptide-based hair conditioners may also be prepared by covalentlyattaching a specific hair-binding peptide to a hair conditioning agent,either directly or through a spacer. Any known peptide or proteinconjugation chemistry may be used to form the peptide-based hairconditioners of the present invention. Conjugation chemistries arewell-known in the art (see for example, Hermanson, BioconjugateTechniques, Academic Press, New York (1996)). Suitable coupling agentsinclude, but are not limited to, carbodiimide coupling agents, diacidchlorides, diisocyanates and other difunctional coupling reagents thatare reactive toward terminal amine and/or carboxylic acid terminalgroups on the peptides and to amine, carboxylic acid, or alcohol groupson the hair conditioning agent. The preferred coupling agents arecarbodiimide coupling agents, such as1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) andN,N′-dicyclohexyl-carbodiimide (DCC), which may be used to activatecarboxylic acid groups for coupling to alcohol, and amine groups.Additionally, it may be necessary to protect reactive amine orcarboxylic acid groups on the peptide to produce the desired structurefor the peptide-based hair conditioner. The use of protecting groups foramino acids, such as t-butyloxycarbonyl (t-Boc), are well known in theart (see for example Stewart et al., supra; Bodanszky, supra; andPennington et al., supra). In some cases it may be necessary tointroduce reactive groups, such as carboxylic acid, alcohol, amine, oraldehyde groups, on the hair conditioning agent for coupling to thehair-binding peptide. These modifications may be done using routinechemistry such as oxidation, reduction and the like, which is well knownin the art.

It may also be desirable to couple the hair-binding peptide to the hairconditioning agent via a spacer. The spacer serves to separate theconditioning agent from the peptide to ensure that the agent does notinterfere with the binding of the peptide to the hair. The spacer may beany of a variety of molecules, such as alkyl chains, phenyl compounds,ethylene glycol, amides, esters and the like. Preferred spacers arehydrophilic and have a chain length from 1 to about 100 atoms, morepreferably, from 2 to about 30 atoms. Examples of preferred spacersinclude, but are not limited to ethanol amine, ethylene glycol,polyethylene with a chain length of 6 carbon atoms, polyethylene glycolwith 3 to 6 repeating units, phenoxyethanol, propanolamide, butyleneglycol, butyleneglycolamide, propyl phenyl chains, and ethyl, propyl,hexyl, steryl, cetyl, and palmitoyl alkyl chains. The spacer may becovalently attached to the peptide and the hair conditioning agent usingany of the coupling chemistries described above. In order to facilitateincorporation of the spacer, a bifunctional cross-linking agent thatcontains a spacer and reactive groups at both ends for coupling to thepeptide and the conditioning agent may be used. Suitable bifunctionalcross-linking agents are well known in the art and include, but are notlimited to diamines, such a as 1,6-diaminohexane; dialdehydes, such asglutaraldehyde; bis N-hydroxysuccinimide esters, such as ethyleneglycol-bis(succinic acid N-hydroxysuccinimide ester), disuccinimidylglutarate, disuccinimidyl suberate, and ethyleneglycol-bis(succinimidylsuccinate); diisocyanates, such ashexamethylenediisocyanate; bis oxiranes, such as 1,4 butanediyldiglycidyl ether; dicarboxylic acids, such as succinyldisalicylate; andthe like. Heterobifunctional cross-linking agents, which contain adifferent reactive group at each end, may also be used. Examples ofheterobifunctional cross-linking agents include, but are not limited tocompounds having the following structure:

where: R₁ is H or a substituent group such as —SO₃Na, —NO₂, or —Br; andR₂ is a spacer such as —CH₂CH₂ (ethyl), —(CH₂)₃ (propyl), or —(CH₂)₃C₆H₅(propyl phenyl). An example of such a heterobifunctional cross-linkingagent is 3-maleimidopropionic acid N-hydroxysuccinimide ester. TheN-hydroxysuccinimide ester group of these reagents reacts with amine oralcohol groups on the conditioner, while the maleimide group reacts withthiol groups present on the peptide. A thiol group may be incorporatedinto the peptide by adding a cysteine group to at least one end of thebinding peptide sequence (i.e., the C-terminus or N-terminus). Severalspacer amino acid residues, such as glycine, may be incorporated betweenthe binding peptide sequence and the terminal cysteine to separate thereacting thiol group from the binding sequence.

Additionally, the spacer may be a peptide composed of any amino acid andmixtures thereof. The preferred peptide spacers are composed of theamino acids glycine, alanine, and serine, and mixtures thereof. Inaddition, the peptide spacer may contain a specific enzyme cleavagesite, such as the protease Caspase 3 site, given by SEQ ID NO:65, whichallows for the enzymatic removal of the conditioning agent from thehair. The peptide spacer may be from 1 to about 50 amino acids,preferably from 1 to about 20 amino acids. These peptide spacers may belinked to the binding peptide sequence by any method known in the art.For example, the entire binding peptide-peptide spacer diblock may beprepared using the standard peptide synthesis methods described supra.In addition, the binding peptide and peptide spacer blocks may becombined using carbodiimide coupling agents (see for example, Hermanson,Bioconjugate Techniques, Academic Press, New York (1996)), diacidchlorides, diisocyanates and other difunctional coupling reagents thatare reactive to terminal amine and/or carboxylic acid terminal groups onthe peptides. Alternatively, the entire binding peptide-peptide spacerdiblock may be prepared using the recombinant DNA and molecular cloningtechniques described supra. The spacer may also be a combination of apeptide spacer and an organic spacer molecule, which may be preparedusing the methods described above.

It may also be desirable to have multiple hair-binding peptides coupledto the hair conditioning agent to enhance the interaction between thepeptide-based hair conditioner and the hair. Either multiple copies ofthe same hair-binding peptide or a combination of different hair-bindingpeptides may be used. In the case of large conditioning particles (e.g.,particle emulsions), a large number of hair-binding peptides, i.e., upto about 1,000, may be coupled to the conditioning agent. A smallernumber of hair-binding peptides can be coupled to the smallerconditioner molecules, i.e., up to about 50. Therefore, in oneembodiment of the present invention, the peptide-based hair conditionersare diblock compositions consisting of a hair-binding peptide (HBP) anda hair conditioning agent (HCA), having the general structure(HBP)_(n)-HCA, where n ranges from 1 to about 1,000, preferably from 1to about 50. In another embodiment, the peptide-based hair conditionerscontain a spacer (S) separating the hair-binding peptide from the hairconditioning agent, as described above. Multiple copies of thehair-binding peptide may be coupled to a single spacer molecule. In thisembodiment, the peptide-based hair conditioners are triblockcompositions consisting of a hair-binding peptide, a spacer, and a hairconditioning agent, having the general structure [(HBP)_(m)-S]_(n)-HCA,where n ranges from 1 to about 1,000, preferably n is 1 to about 50, andm ranges from 1 to about 50, preferably m is 1 to about 10.

It should be understood that as used herein, HBP is a genericdesignation and is not meant to refer to a single hair binding peptidesequence. Where n or m as used above, is greater than 1, it is wellwithin the scope of the invention to provide for the situation where aseries of hair binding peptides of different sequences may form a partof the composition. Additionally, it should be understood that thesestructures do not necessarily represent a covalent bond between thepeptide, the hair conditioning agent, and the optional spacer. Asdescribed above, the coupling interaction between the peptide, the hairconditioning agent, and the optional spacer may be either covalent ornon-covalent.

The peptide-based hair conditioners may be used in compositions for haircare. It should also be recognized that the hair-binding peptidesthemselves can serve as conditioning agents for the treatment of hair.Hair care compositions are herein defined as compositions for thetreatment of hair, including but not limited to shampoos, conditioners,lotions, aerosols, gels, mousses, and hair dyes comprising an effectiveamount of a peptide-based hair conditioner or a mixture of differentpeptide-based hair conditioners in a cosmetically acceptable medium. Aneffective amount of a peptide-based hair conditioner or hair-bindingpeptide for use in a hair care composition is herein defined as aproportion of from about 0.01% to about 10%, preferably about 0.01% toabout 5% by weight relative to the total weight of the composition.Components of a cosmetically acceptable medium for hair carecompositions are described by Philippe et al. in U.S. Pat. No.6,280,747, and by Omura et al. in U.S. Pat. No. 6,139,851 and Cannell etal. in U.S. Pat. No. 6,013,250, all of which are incorporated herein byreference. For example, these hair care compositions can be aqueous,alcoholic or aqueous-alcoholic solutions, the alcohol preferably beingethanol or isopropanol, in a proportion of from about 1 to about 75% byweight relative to the total weight, for the aqueous-alcoholicsolutions. Additionally, the hair care compositions may contain one ormore conventional cosmetic or dermatological additives or adjuvantsincluding but not limited to, antioxidants, preserving agents, fillers,surfactants, UVA and/or UVB sunscreens, fragrances, thickeners, wettingagents and anionic, nonionic or amphoteric polymers, and dyes orpigments.

Peptide-Based Skin Conditioners

The peptide-based skin conditioners are formed by coupling askin-binding peptide (SBP) with a skin conditioning agent (SCA). Theskin-binding peptide part of the conditioner binds strongly to the skin,thus keeping the conditioning agent attached to the skin for a longlasting conditioning effect. The skin-binding peptides include, but arenot limited to, skin-binding peptides selected by the screening methodsdescribed above, including the skin-binding peptide sequence of theinvention, given as SEQ ID NO:61. Additionally, any known skin-bindingpeptide may be used, including but not limited to SEQ ID NO:2, and SEQID NOs:99-104, described by Janssen et al. in U.S. Patent ApplicationPublication No. 2003/0152976 and by Janssen et al. in WO 04048399,respectively.

Skin conditioning agents as herein defined include, but are not limitedto astringents, which tighten skin; exfoliants, which remove dead skincells; emollients, which help maintain a smooth, soft, pliableappearance; humectants, which increase the water content of the toplayer of skin; occlusives, which retard evaporation of water from theskin's surface; and miscellaneous compounds that enhance the appearanceof dry or damaged skin or reduce flaking and restore suppleness. In thepeptide-based skin conditioners, any known skin conditioning agent maybe used. Skin conditioning agents are well known in the art, see forexample Green et al. (WO 0107009), and are available commercially fromvarious sources. Suitable examples of skin conditioning agents include,but are not limited to, alpha-hydroxy acids, beta-hydroxy acids,polyols, hyaluronic acid, D,L-panthenol, polysalicylates, vitamin Apalmitate, vitamin E acetate, glycerin, sorbitol, silicones, siliconederivatives, lanolin, natural oils and triglyceride esters. Thepreferred skin conditioning agents are polysalicylates, propylene glycol(CAS No. 57-55-6, Dow Chemical, Midland, Mich.), glycerin (CAS No.56-81-5, Proctor & Gamble Co., Cincinnati, Ohio), glycolic acid (CAS No.79-14-1, DuPont Co., Wilmington, Del.), lactic acid (CAS No. 50-21-5,Alfa Aesar, Ward Hill, Mass.), malic acid (CAS No. 617-48-1, AlfaAesar), citric acid (CAS No. 77-92-9, Alfa Aesar), tartaric acid (CASNO. 133-37-9, Alfa Aesar), glucaric acid (CAS No. 87-73-0), galactaricacid (CAS No. 526-99-8), 3-hydroxyvaleric acid (CAS No. 10237-77-1),salicylic acid (CAS No. 69-72-7, Alfa Aesar), and 1,3 propanediol (CASNo. 504-63-2, DuPont Co., Wilmington, Del.). Polysalicylates may beprepared by the method described by White et al. in U.S. Pat. No.4,855,483, incorporated herein by reference. Glucaric acid may besynthesized using the method described by Merbouh et al. (Carbohydr.Res. 336:75-78 (2001). The 3-hydroxyvaleric acid may be prepared asdescribed by Bramucci in WO 02012530.

The peptide-based skin conditioners are prepared by coupling a specificskin-binding peptide to the skin conditioning agent, either directly orvia a spacer. Any of the coupling methods described above may be used.It may be necessary to introduce reactive groups, such as carboxylicacid, alcohol, amine, or aldehyde groups, on the skin conditioning agentfor coupling to the hair-binding peptide, as described above. It mayalso be desirable to have multiple skin-binding peptides coupled to theskin conditioning agent to enhance the interaction between thepeptide-based skin conditioner and the skin. Either multiple copies ofthe same skin-binding peptide or a combination of different skin-bindingpeptides may be used. In the case of large conditioning particles, alarge number of skin-binding peptides, i.e., up to about 1,000, may becoupled to the conditioning agent. A smaller number of skin-bindingpeptides can be attached to the smaller conditioner molecules, i.e., upto about 50. Therefore, in one embodiment, the peptide-based skinconditioners are diblock compositions consisting of a skin-bindingpeptide (SBP) and a skin conditioning agent (SCA), having the generalstructure (SBP)_(n)-SCA, where n ranges from 1 to about 1,000,preferably from 1 to about 50.

In another embodiment, the peptide-based skin conditioners contain aspacer (S) separating the skin-binding peptide from the skinconditioning agent, as described above. Multiple copies of theskin-binding peptide may be coupled to a single spacer molecule. In thisembodiment, the peptide-based skin conditioners are triblockcompositions consisting of a skin binding peptide, a spacer, and a skinconditioning agent, having the general structure [(SBP)_(m)-S]_(n)-SCA,where n ranges from 1 to about 1,000, preferably n is 1 to about 50, andm ranges from 1 to about 50, preferably m is 1 to about 10.

It should be understood that as used herein, SBP is a genericdesignation and is not meant to refer to a single skin binding peptidesequence. Where n or m as used above, is greater than 1, it is wellwithin the scope of the invention to provide for the situation where aseries of skin binding peptides of different sequences may form a partof the composition. Additionally, it should be understood that thesestructures do not necessarily represent a covalent bond between thepeptide, the skin conditioning agent, and the optional spacer. Asdescribed above, the coupling interaction between the peptide, the skinconditioning agent, and the optional spacer may be either covalent ornon-covalent.

The peptide-based skin conditioners may be used in compositions for skincare. It should also be recognized that the skin-binding peptidesthemselves can serve as conditioning agents for skin. Skin carecompositions are herein defined as compositions comprising an effectiveamount of a peptide-based skin conditioner or a mixture of differentpeptide-based skin conditioners in a cosmetically acceptable medium. Theuses of these compositions include, but are not limited to, skin care,skin cleansing, make-up, and anti-wrinkle products. An effective amountof a peptide-based skin conditioner or skin-binding peptide for skincare compositions is herein defined as a proportion of from about 0.001%to about 10%, preferably about 0.01% to about 5% by weight relative tothe total weight of the composition. This proportion may vary as afunction of the type of skin care composition. Suitable compositions fora cosmetically acceptable medium are described by Philippe et al. supra.For example, the cosmetically acceptable medium may be an anhydrouscomposition containing a fatty substance in a proportion generally offrom about 10 to about 90% by weight relative to the total weight of thecomposition, where the fatty phase containing at least one liquid, solidor semi-solid fatty substance. The fatty substance includes, but is notlimited to, oils, waxes, gums, and so-called pasty fatty substances.Alternatively, the compositions may be in the form of a stabledispersion such as a water-in-oil or oil-in-water emulsion.Additionally, the compositions may contain one or more conventionalcosmetic or dermatological additives or adjuvants, including but notlimited to, antioxidants, preserving agents, fillers, surfactants, UVAand/or UVB sunscreens, fragrances, thickeners, wetting agents andanionic, nonionic or amphoteric polymers, and dyes or pigments.

Peptide-Based Hair Colorants

The peptide-based hair colorants are formed by coupling a hair-bindingpeptide (HBP) with a coloring agent (C). The hair-binding peptide partof the peptide-based hair colorant binds strongly to the hair, thuskeeping the coloring agent attached to the hair for a long lasting haircoloring effect. The hair-binding peptides include, but are not limitedto, hair-binding peptides selected by the screening methods describedabove, including the hair-binding peptide sequences of the invention,given by SEQ ID NOs: 3-59, 64, 66, 69 and 70, most preferably thepeptides given by SEQ ID NO:46 and SEQ ID NO:66, which bind strongly tohair, but not to skin. Additionally, any known hair-binding peptide maybe used, including but not limited to SEQ ID NO:1, and SEQ ID NOs:76-98,described by Janssen et al. in U.S. Patent Application Publication No.2003/0152976 and by Janssen et al. in WO 04048399, respectively. Forbleached hair, the fingernail-binding peptide, given as SEQ ID NO:60,may also be used.

Coloring agents as herein defined are any dye, pigment, and the likethat may be used to change the color of hair, skin, or nails. In thepeptide-based hair colorants, any known coloring agent may be used. Haircoloring agents are well known in the art (see for example Green et al.supra, CFTA International Color Handbook, 2^(nd) ed., Micelle Press,England (1992) and Cosmetic Handbook, US Food and Drug Administration,FDA/IAS Booklet (1992)), and are available commercially from varioussources (for example Bayer, Pittsburgh, Pa.; Ciba-Geigy, Tarrytown,N.Y.; ICI, Bridgewater, N.J.; Sandoz, Vienna, Austria; BASF, MountOlive, N.J.; and Hoechst, Frankfurt, Germany). Suitable hair coloringagents include, but are not limited to dyes, such as4-hydroxypropylamino-3-nitrophenol, 4-amino-3-nitrophenol,2-amino-6-chloro-4-nitrophenol, 2-nitro-paraphenylenediamine,N,N-hydroxyethyl-2-nitro-phenylenediamine, 4-nitro-indole, Henna, HCBlue 1, HC Blue 2, HC Yellow 4, HC Red 3, HC Red 5, Disperse Violet 4,Disperse Black 9, HC Blue 7, HC Blue 12, HC Yellow 2, HC Yellow 6, HCYellow 8, HC Yellow 12, HC Brown 2, D&C Yellow 1, D&C Yellow 3, D&C Blue1, Disperse Blue 3, Disperse violet 1, eosin derivatives such as D&C RedNo. 21 and halogenated fluorescein derivatives such as D&C Red No. 27,D&C Red Orange No. 5 in combination with D&C Red No. 21 and D&C OrangeNo. 10; and pigments, such as D&C Red No. 36 and D&C Orange No. 17, thecalcium lakes of D&C Red Nos. 7, 11, 31 and 34, the barium lake of D&CRed No. 12, the strontium lake of D&C Red No. 13, the aluminum lakes ofFD&C Yellow No. 5, of FD&C Yellow No. 6, of D&C Red No. 27, of D&C RedNo. 21, and of FD&C Blue No. 1, iron oxides, manganese violet, chromiumoxide, titanium dioxide, titanium dioxide nanoparticles, zinc oxide,barium oxide, ultramarine blue, bismuth citrate, and carbon blackparticles. The preferred hair coloring agents are D&C Yellow 1 and 3, HCYellow 6 and 8, D&C Blue 1, HC Blue 1, HC Brown 2, HC Red5,2-nitro-paraphenylenediamine,N,N-hydroxyethyl-2-nitro-phenylenediamine, 4-nitro-indole, and carbonblack.

Metallic and semiconductor nanoparticles may also be used as haircoloring agents due to their strong emission of light (Vic et al. U.S.Patent Application Publication No. 2004/0010864). The metallicnanoparticles include, but are not limited to, particles of gold,silver, platinum, palladium, iridium, rhodium, osmium, iron, copper,cobalt, and alloys composed of these metals. An “alloy” is hereindefined as a homogeneous mixture of two or more metals. The“semiconductor nanoparticles” include, but are not limited to, particlesof cadmium selenide, cadmium sulfide, silver sulfide, cadmium sulfide,zinc oxide, zinc sulfide, zinc selenide, lead sulfide, gallium arsenide,silicon, tin oxide, iron oxide, and indium phosphide. The nanoparticlesare stabilized and made water-soluble by the use of a suitable organiccoating or monolayer. As used herein, monolayer-protected nanoparticlesare one type of stabilized nanoparticle. Methods for the preparation ofstabilized, water-soluble metal and semiconductor nanoparticles areknown in the art, and are described by Huang et al. in U.S. patentapplication Ser. No. 10/622,889, which is incorporated herein byreference. The color of the nanoparticles depends on the size of theparticles. Therefore, by controlling the size of the nanoparticles,different colors may be obtained. For example, ZnS-coated CdSenanoparticles cover the entire visible spectrum over a particle sizerange of 2 to 6 nm. Specifically, CdSe nanoparticles with a core size of2.3, 4.2, 4.8 and 5.5 nm emit light at the wavelength centered around485, 565, 590, and 625 nm, respectively. Water-soluble nanoparticles ofdifferent sizes may be obtained from a broad size distribution ofnanoparticles using the size fractionation method described by Huang,supra. That method comprises the regulated addition of a water-miscibleorganic solvent to a solution of nanoparticles in the presence of anelectrolyte. Increasing additions of the water-miscible organic solventresult in the precipitation of nanoparticles of decreasing size. Themetallic and semiconductor nanoparticles may also serve as volumizingagents, as described above.

Of particular utility are titanium dioxide nanoparticles that not onlyserve as a colorant but additionally may serve to block harmful UVradiation. Suitable titanium dioxide nanoparticles are described in U.S.Pat. Nos. 5,451,390; 5,672,330; and 5,762,914. Titanium dioxide P25 isan example of a suitable commercial product available from Degussa.Other commercial suppliers of titanium dioxide nanoparticles includeKemira, Sachtleben and Tayca.

The titanium dioxide nanoparticles typically have an average particlesize diameter of less than 100 nanometers (nm) as determined by dynamiclight scattering which measures the particle size distribution ofparticles in liquid suspension. The particles are typically agglomerateswhich may range from about 3 nm to about 6000 nm. Any process known inthe art can be used to prepare such particles. The process may involvevapor phase oxidation of titanium halides or solution precipitation fromsoluble titanium complexes, provided that titanium dioxide nanoparticlesare produced.

A preferred process to prepare titanium dioxide nanoparticles is byinjecting oxygen and titanium halide, preferably titanium tetrachloride,into a high-temperature reaction zone, typically ranging from 400 to2000 degrees centigrade. Under the high temperature conditions presentin the reaction zone, nanoparticles of titanium dioxide are formedhaving high surface area and a narrow size distribution. The energysource in the reactor may be any heating source such as a plasma torch.

Additionally, the coloring agent may be a colored, polymericmicrosphere. Exemplary polymeric microspheres include, but are notlimited to, microspheres of polystyrene, polymethylmethacrylate,polyvinyltoluene, styrene/butadiene copolymer, and latex. For use in theinvention, the microspheres have a diameter of about 10 nanometers toabout 2 microns. The microspheres may be colored by coupling anysuitable dye, such as those described above, to the microspheres. Thedyes may be coupled to the surface of the microsphere or adsorbed withinthe porous structure of a porous microsphere. Suitable microspheres,including undyed and dyed microspheres that are functionalized to enablecovalent attachment, are available from companies such as BangLaboratories (Fishers, Ind.).

The peptide-based hair colorants are prepared by coupling a specifichair-binding peptide to a coloring agent, either directly or via aspacer. Any of the coupling methods described above may be used. It maybe necessary to introduce reactive groups, such as carboxylic acid,alcohol, amine, or aldehyde groups, on the coloring agent for couplingto the hair-binding peptide. These modifications may be done usingroutine chemistry, which is well known in the art. For example, thesurface of carbon black particles may be oxidized using nitric acid, aperoxide such as hydrogen peroxide, or an inorganic initiator such asammonium persulfate, to generate functional groups. Preferably, thecarbon black surface is oxidized using ammonium persulfate as describedby Carrasco-Marin et al. (J. Chem. Soc., Faraday Trans. 93:2211-2215(1997)). Amino functional groups may be introduced to the surface ofcarbon black using an organic initiator such as2,2′-Azobis(2-methylpropionamide)-dihydrochloride. The inorganicpigments and the nanoparticles may be derivatized to introducecarboxylic acid or amino functional groups in a similar manner.

Additionally, the hair-binding peptide may be coupled to a pigment usinga pigment-binding peptide. Suitable pigment-binding peptide sequencesare known in the art. For example, Nomoto et al. in EP1275728 describepeptides that bind to carbon black, copper phthalocyanine, titaniumdioxide, and silicon dioxide. O'Brien et al. in co-pending and commonlyowned U.S. patent application Ser. No. 10/935,254 describe peptides thatbind to carbon black, Cromophtal® Yellow, Sunfast® Magenta, and Sunfast®Blue. Additional pigment-binding peptides may be identified using theany of the screening methods described above. The pigment-bindingpeptide may be coupled to the hair-binding peptide either directly orthrough a spacer using any of the coupling methods described above. Thehair-binding peptide-pigment binding peptide diblock or triblock (if aspacer is used) is contacted with the pigment to attach it to thepigment-binding peptide.

It may also be desirable to have multiple hair-binding peptides coupledto the coloring agent to enhance the interaction between thepeptide-based hair colorant and the hair. Either multiple copies of thesame hair-binding peptide or a combination of different hair-bindingpeptides may be used. In the case of large pigment particles, a largenumber of hair-binding peptides, i.e., up to about 10,000, may becoupled to the pigment. A smaller number of hair-binding peptides can becoupled to the smaller dye molecules, i.e., up to about 50. Therefore,in one embodiment, the peptide-based hair colorants are diblockcompositions consisting of a hair-binding peptide (HBP) and a coloringagent (C), having the general structure (HBP)_(n)-C, where n ranges from1 to about 10,000, preferably n is 1 to about 500.

In another embodiment, the peptide-based hair colorants contain a spacer(S) separating the binding peptide from the hair coloring agent, asdescribed above. Multiple copies of the hair-binding peptide may becoupled to a single spacer molecule. In this embodiment, thepeptide-based hair colorants are triblock compositions consisting of ahair-binding peptide, a spacer, and a coloring agent, having the generalstructure [(HBP)_(m)-S]_(n)-C, where n ranges from 1 to about 10,000,preferably n is 1 to about 500, and m ranges from 1 to about 50,preferably m is 1 to about 10.

It should be understood that as used herein, HBP is a genericdesignation and is not meant to refer to a single hair binding peptidesequence. Where n or m as used above, is greater than 1, it is wellwithin the scope of the invention to provide for the situation where aseries of hair binding peptides of different sequences may form a partof the composition. Additionally, it should be understood that thesestructures do not necessarily represent a covalent bond between thepeptide, the coloring agent, and the optional spacer. As describedabove, the coupling interaction between the peptide, the coloring agent,and the optional spacer may be either covalent or non-covalent.

The peptide-based hair colorants may be used in hair coloringcompositions for dyeing hair. Hair coloring compositions are hereindefined as compositions for the coloring, dyeing, or bleaching of hair,comprising an effective amount of peptide-based hair colorant or amixture of different peptide-based hair colorants in a cosmeticallyacceptable medium. An effective amount of a peptide-based hair colorantfor use in a hair coloring composition is herein defined as a proportionof from about 0.001% to about 20% by weight relative to the total weightof the composition. Components of a cosmetically acceptable medium forhair coloring compositions are described by Dias et al., in U.S. Pat.No. 6,398,821 and by Deutz et al., in U.S. Pat. No. 6,129,770, both ofwhich are incorporated herein by reference. For example, hair coloringcompositions may contain sequestrants, stabilizers, thickeners, buffers,carriers, surfactants, solvents, antioxidants, polymers, andconditioners. The conditioners may include the peptide-based hairconditioners and hair-binding peptides in a proportion from about 0.01%to about 10%, preferably about 0.01% to about 5% by weight relative tothe total weight of the hair coloring composition.

The peptide-based hair colorants may also be used as coloring agents incosmetic compositions that are applied to the eyelashes or eyebrowsincluding, but not limited to mascaras, and eyebrow pencils. These maybe anhydrous make-up products comprising a cosmetically acceptablemedium which contains a fatty substance in a proportion generally offrom about 10 to about 90% by weight relative to the total weight of thecomposition, where the fatty phase containing at least one liquid, solidor semi-solid fatty substance, as described above. The fatty substanceincludes, but is not limited to, oils, waxes, gums, and so-called pastyfatty substances. Alternatively, these compositions may be in the formof a stable dispersion such as a water-in-oil or oil-in-water emulsion,as described above. In these compositions, the proportion of thepeptide-based hair colorant is generally from about 0.001% to about 20%by weight relative to the total weight of the composition.

Peptide-Based Nail Colorants

The peptide-based nail colorants are formed by coupling a nail-bindingpeptide (NBP) with a coloring agent (C). The nail-binding peptide partof the peptide-based nail colorant binds strongly to the fingernails ortoenails, thus keeping the coloring agent attached to the nails for along lasting coloring effect. The nail-binding peptides include, but arenot limited to nail-binding peptides selected by the screening methodsdescribed above, including the nail-binding peptide sequences of theinvention, given by SEQ ID NOs:53 and 60, most preferably the peptidegiven by SEQ ID NO:60. Additionally, the beached hair-binding peptides,given as SEQ ID NOs:7, 8, 19-27 38, 39, 40, 43-45, 47, 57, 58. and 59may be used.

The peptide-based nail colorants are prepared by coupling a specificnail-binding peptide to a coloring agent, either directly or via aspacer, using any of the coupling methods described above. In thepeptide-based nail colorants, any of the coloring agents described abovemay be used. The preferred coloring agents for use in the peptide-basednail colorants include D&C Red Nos. 8, 10, 30 and 36, the barium lakesof D&C Red Nos. 6, 9 and 12, the calcium lakes of D&C Red Nos. 7, 11, 31and 34, the strontium lake of D&C Red No. 30 and D&C Orange No. 17 andD&C Blue No. 6.

It may also be desirable to have multiple nail-binding peptides coupledto the coloring agent to enhance the interaction between thepeptide-based nail colorant and the nails. Either multiple copies of thesame nail-binding peptide or a combination of different nail-bindingpeptides may be used. In the case of large pigment particles, a largenumber of nail-binding peptides, i.e., up to about 10,000, may becoupled to the pigment. A smaller number of nail-binding peptides can becoupled to the smaller dye molecules, i.e., up to about 50. Therefore,in one embodiment, the peptide-based nail colorants are diblockcompositions consisting of a nail-binding peptide (NBP) and a coloringagent (C), having the general structure (NBP)_(n)-C, where n ranges from1 to about 10,000, preferably n is 1 to about 500.

In another embodiment, the peptide-based nail colorants contain a spacer(S) separating the binding peptide from the coloring agent, as describedabove. Multiple copies of the nail-binding peptide may be coupled to asingle spacer molecule. In this embodiment, the peptide-based nailcolorants are triblock compositions consisting of a nail-bindingpeptide, a spacer, and a coloring agent, having the general structure[(NBP)_(m)-S]_(n)-C, where n ranges from 1 to about 10,000, preferably nis 1 to about 500, and m ranges from 1 to about 50, preferably m is 1 toabout 10.

It should be understood that as used herein, NBP is a genericdesignation and is not meant to refer to a single nail binding peptidesequence. Where n or m as used above, is greater than 1, it is wellwithin the scope of the invention to provide for the situation where aseries of nail binding peptides of different sequences may form a partof the composition. Additionally, it should be understood that thesestructures do not necessarily represent a covalent bond between thepeptide, the coloring agent, and the optional spacer. As describedabove, the coupling interaction between the peptide, the coloring agent,and the optional spacer may be either covalent or non-covalent.

The peptide-based nail colorants may be used in nail polish compositionsfor coloring fingernails and toenails. Nail polish compositions areherein defined as compositions for the treatment and coloring of nails,comprising an effective amount of a peptide-based nail colorant or amixture of different peptide-based nail colorants in a cosmeticallyacceptable medium. An effective amount of a peptide-based nail colorantfor use in a nail polish composition is herein defined as a proportionof from about 0.001% to about 20% by weight relative to the total weightof the composition. Components of a cosmetically acceptable medium fornail polishes are described by Philippe et al. supra. The nail polishcomposition typically contains a solvent and a film forming substance,such as cellulose derivatives, polyvinyl derivatives, acrylic polymersor copolymers, vinyl copolymers and polyester polymers. Additionally,the nail polish may contain a plasticizer, such as tricresyl phosphate,benzyl benzoate, tributyl phosphate, butyl acetyl ricinoleate, triethylcitrate, tributyl acetyl citrate, dibutyl phthalate or camphor.

Peptide-Based Skin Colorants

The peptide-based skin colorants are formed by coupling a skin-bindingpeptide (SBP) with a coloring agent (C). The skin-binding peptide partof the peptide-based skin colorant binds strongly to the skin, thuskeeping the coloring agent attached to the skin for a long lasting skincoloring effect. The skin-binding peptides include, but are not limitedto, skin-binding peptides selected by the screening methods describedabove, including the skin-binding peptide sequence of the invention,given as SEQ ID NOs:61. Additionally, any known skin-binding peptide maybe used, including but not limited to SEQ ID NO:2, and SEQ IDNOs:99-104, described by Janssen et al. in U.S. Patent ApplicationPublication No. 2003/0152976 and by Janssen et al. in WO 04048399,respectively.

The peptide-based skin colorants are prepared by coupling a specificskin-binding peptide to a coloring agent, either directly or via aspacer, using any of the coupling methods described above. Any of thecolorants described above may be used. The preferred coloring agents foruse in the peptide-based skin colorants include the following dyes:eosin derivatives such as D&C Red No. 21 and halogenated fluoresceinderivatives such as D&C Red No. 27, D&C Red Orange No. 5 in combinationwith D&C Red No. 21 and D&C Orange No. 10, and the pigments: titaniumdioxide, titanium dioxide nanoparticles, zinc oxide, D&C Red No. 36 andD&C Orange No. 17, the calcium lakes of D&C Red Nos. 7, 11, 31 and 34,the barium lake of D&C Red No. 12, the strontium lake D&C Red No. 13,the aluminum lakes of FD&C Yellow No. 5, of FD&C Yellow No. 6, of D&CRed No. 27, of D&C Red No. 21, of FD&C Blue No. 1, iron oxides,manganese violet, chromium oxide, ultramarine blue, and carbon black.The coloring agent may also be a sunless tanning agent, such asdihydroxyacetone, that produces a tanned appearance on the skin withoutexposure to the sun.

It may also be desirable to have multiple skin-binding peptides coupledto the coloring agent to enhance the interaction between thepeptide-based skin colorant and the skin. Either multiple copies of thesame skin-binding peptide or a combination of different skin-bindingpeptides may be used. In the case of large pigment particles, a largenumber of skin-binding peptides, i.e., up to about 10,000, may becoupled to the pigment. A smaller number of skin-binding peptides can becoupled to the smaller dye molecules, i.e., up to about 50. Therefore,in one embodiment, the peptide-based skin colorants are diblockcompositions consisting of a skin-binding peptide (SBP) and a coloringagent (C), having the general structure (SBP)_(n)-C, where n ranges from1 to about 10,000, preferably n is 1 to about 500.

In another embodiment, the peptide-based skin colorants contain a spacer(S) separating the binding peptide from the coloring agent, as describedabove. Multiple copies of the skin-binding peptide may be coupled to asingle spacer molecule. In this embodiment, the peptide-based skincolorants are triblock compositions consisting of a skin-bindingpeptide, a spacer, and a coloring agent, having the general structure[(SBP)_(m)-S]_(n)-C, where n ranges from 1 to about 10,000, preferably nis 1 to about 500, and m ranges from 1 to about 50, preferably m is 1 toabout 10.

It should be understood that as used herein, SBP is a genericdesignation and is not meant to refer to a single skin binding peptidesequence. Where n or m as used above, is greater than 1, it is wellwithin the scope of the invention to provide for the situation where aseries of skin binding peptides of different sequences may form a partof the composition. Additionally, It should be understood that thesestructures do not necessarily represent a covalent bond between thepeptide, the coloring agent, and the optional spacer. As describedabove, the coupling interaction between the peptide, the coloring agent,and the optional spacer may be either covalent or non-covalent.

The peptide-based skin colorants may be used as coloring agents incosmetic and make-up products, including but not limited to foundations,blushes, lipsticks, lip liners, lip glosses, eyeshadows and eyeliners.These may be anhydrous make-up products comprising a cosmeticallyacceptable medium which contains a fatty substance, or they may be inthe form of a stable dispersion such as a water-in-oil or oil-in-wateremulsion, as described above. In these compositions, the proportion ofthe peptide-based skin colorant is generally from about 0.001% to about40% by weight relative to the total weight of the composition.

Peptide-Based Oral Care Reagents

The peptide-based oral care reagents of the invention are formed bycoupling an oral cavity surface-binding peptide (OBP) with an oral carebenefit agent (OBA). Oral cavity surface-binding peptides include, butare not limited to, tooth-binding peptides (TBP), skin-binding peptides(SBP), gum, cheek, and tongue-binding peptides. The peptide part of thepeptide-based oral care agent binds strongly to the teeth, gums, cheeks,tongue, or other surface in the oral cavity, thus keeping the benefitagent attached for a long lasting effect. The skin-binding peptidesdescribed above may be useful for attachment to gums, cheeks, or tongue.Preferably, a binding peptide for the specific oral cavity surface ofinterest is identified using the screening methods described above.Preferred oral cavity surface-binding peptides of the invention arethose that bind to teeth. The term “tooth surface” will refer to asurface comprised of tooth enamel (typically exposed after professionalcleaning or polishing) or tooth pellicle (an acquired surface comprisingsalivary glycoproteins). Hydroxyapatite can be coated with salivaryglycoproteins to mimic a natural tooth pellicle surface (tooth enamel ispredominantly comprised of hydroxyapatite). As used herein, the terms“pellicle” and “tooth pellicle” will refer to the thin film (typicallyranging from about 1 μm to about 200 μm thick) derived from salivaryglycoproteins which forms over the surface of the tooth crown. Dailytooth brushing tends to only remove a portion of the pellicle surfacewhile abrasive tooth cleaning and/or polishing (typically by a dentalprofessional) will expose more of the tooth enamel surface. As usedherein, the terms “enamel” and “tooth enamel” will refer to the highlymineralized tissue which forms the outer layer of the tooth. The enamellayer is composed primarily of crystalline calcium phosphate (i.e.hydroxyapatite) along with water and some organic material. In oneembodiment, the tooth surface is selected from the group consisting oftooth enamel and tooth pellicle. As used herein, the term “tooth-bindingpeptide” will refer to a peptide that binds to tooth enamel or toothpellicle. Tooth binding peptides, therefore, fall into two generalcategories, those that bind the pellicle coating of the tooth and thosethat binding directly to the tooth enamel. Examples of toothpellicle-binding peptides are those having the amino acid sequences asset forth in SEQ ID NOs: 106-125. Examples of tooth enamel-bindingpeptides are those having the amino acid sequences as set forth in SEQID NOs: 126-145.

The peptide-based oral care reagents of the invention are prepared bycoupling an oral cavity surface-binding peptide to an oral care benefitagent, either directly or via a spacer, using any of the couplingmethods described above. Oral care benefit agents are well known in theart (see for example White et al., U.S. Pat. No. 6,740,311; Lawler etal., U.S. Pat. No. 6,706,256; and Fuglsang et al., U.S. Pat. No.6,264,925; all of which are incorporated herein by reference). Exemplaryoral benefit agents include, but are not limited to, white colorants,whitening agents, enzymes, anti-plaque agents, anti-staining agents,anti-microbial agents, anti-caries agents, flavoring agents, coolants,and salivating agents.

Suitable white colorants which may be used in peptide-based teethwhiteners include, but are not limited to, white pigments such astitanium dioxide, titanium dioxide nanoparticles; and white mineralssuch as hydroxyapatite, and Zircon (zirconium silicate). Suitableenzymes may be naturally occurring or recombinant enzymes including, butnot limited to, oxidases, peroxidases, proteases, lipases, glycosidases,esterases, and polysaccharide hydrolases. Anti-plaque agents include,but are not limited to, fluoride ion sources and anti-microbial agents.Suitable anti-microbial agents include, but are not limited to,anti-microbial peptides such as those described by Haynie in U.S. Pat.No. 5,847,047, magainins, and cecropins; microbiocides such astriclosan, chlorhexidine, quaternary ammonium compounds, chloroxylenol,chloroxyethanol, phthalic acid and its salts, and thymol. Suitableflavoring agents include, but are not limited to, oil of wintergreen,oil of peppermint, oil of spearmint, menthol, methyl salicylate,eucalyptol, and vanillin.

It may also be desirable to have multiple oral cavity surface-bindingpeptides coupled to the oral benefit agent to enhance the interactionbetween the peptide-based oral care agent and the oral cavity surface.Either multiple copies of the same oral cavity surface-binding peptideor a combination of different oral cavity surface-binding peptides maybe used. In the case of large pigment particles, a large number of oralcavity surface-binding peptides, i.e., up to about 10,000, may becoupled to the pigment. A smaller number of oral cavity surface-bindingpeptides can be coupled to the smaller oral benefit agents molecules,i.e., up to about 50. Therefore, in one embodiment of the presentinvention, the peptide-based oral care reagents are diblock compositionsconsisting of an oral cavity surface-binding peptide (OBP) and an oralbenefit agent (OBA), having the general structure (OBP)_(n)-OBA, where nranges from 1 to about 10,000, preferably n is 1 to about 500.

In another embodiment, the peptide-based oral care reagents contain aspacer (S) separating the binding peptide from the oral benefit agent,as described above. Multiple copies of the oral cavity surface-bindingpeptide may be coupled to a single spacer molecule. In this embodiment,the peptide-based oral care reagents are triblock compositionsconsisting of an oral cavity surface-binding peptide, a spacer, and anoral benefit agent, having the general structure [(OBP)_(m)-S]_(n)-OBA,where n ranges from 1 to about 10,000, preferably n is 1 to about 500,and m ranges from 1 to about 50, preferably m is 1 to about 10.

It should be understood that as used herein, OBP is a genericdesignation and is not meant to refer to a single oral cavitysurface-binding peptide sequence. Where n or m as used above, is greaterthan 1, it is well within the scope of the invention to provide for thesituation where a series of oral cavity surface-binding peptides ofdifferent sequences may form a part of the composition. Additionally, itshould be understood that these structures do not necessarily representa covalent bond between the peptide, the oral benefit agent, and theoptional spacer. As described above, the coupling interaction betweenthe peptide, the oral benefit agent, and the optional spacer may beeither covalent or non-covalent.

The peptide-based oral care reagents of the invention may be used inoral care products, which may have any suitable physical form, such aspowder, paste, gel, liquid, ointment, or tablet. Exemplary oral careproducts include, but are not limited to, toothpaste, dental cream, gelor tooth powder, mouth wash, breath freshener, and dental floss. Theoral care products comprise an effective amount of the peptide-basedoral care reagents of the invention in an orally acceptable carriermedium. An effective amount of a peptide-based oral care agent for usein an oral care product may vary depending on the type of product.Typically, the effective amount of the peptide-based oral care agent isa proportion from about 0.001% to about 90% by weight of the totalproduct composition. The oral care product may contain one type ofpeptide-based oral care agent or a mixture of different peptide-basedoral care reagents.

Components of an orally acceptable carrier medium are described by Whiteet al., Lawler et al., and Fuglsang et al., supra. For example, inaddition to the peptide-based oral care reagents of the invention, theoral care products may contain one or more of the following: abrasives,surfactants, chelating agents, fluoride sources, thickening agents,buffering agents, solvents, humectants, carriers, bulking agents, andadditional oral benefit agents, as given above.

The oral care products of the invention may be prepared using standardtechniques that are well known in the art. If the composition comprisesmore than one phase, typically, the different phases are preparedseparately, with material of similar phase partitioning being added inany order. The two phases are combined using vigorous mixing to form themultiphase system (e.g., an emulsion or dispersion).

Methods for Treating Hair, Skin, Nails, and the Oral Cavity

In another embodiment, methods are provided for treating hair, skin, andnails, with the peptide-based conditioners and colorants of the presentinvention. Specifically, the present invention also comprises a methodfor forming a protective film of peptide-based conditioner on skin,hair, or lips by applying one of the compositions described abovecomprising an effective amount of a peptide-based skin conditioner orpeptide-based hair conditioner to the skin, hair, or lips and allowingthe formation of the protective film. The compositions may be applied tothe skin, hair, or lips by various means, including, but not limited tospraying, brushing, and applying by hand. The peptide-based conditionercomposition is left in contact with the skin, hair, or lips for a periodof time sufficient to form the protective film, preferably for at leastabout 0.1 to 60 min.

The present invention also provides a method for coloring hair byapplying a hair coloring composition comprising an effective amount of apeptide-based hair colorant to the hair by means described above. Thehair coloring composition is allowed to contact the hair for a period oftime sufficient to cause coloration of the hair, preferably betweenabout 5 seconds to about 50 minutes, and more preferably from about 5seconds to about 60 seconds, and then the hair coloring composition maybe rinsed from the hair.

The present invention also provides a method for coloring skin or lipsby applying a skin coloring composition comprising an effective amountof a peptide-based skin colorant to the skin or lips by means describedabove.

The present invention also provides a method for coloring fingernails ortoenails by applying a nail polish composition comprising an effectiveamount of a peptide-based nail colorant to the fingernails or toenailsby means described above.

The present invention also provides a method for coloring eyebrows andeyelashes by applying a cosmetic composition comprising an effectiveamount of a peptide-based hair colorant to the eyebrows and eyelashes bymeans described above.

The invention also provides a method for whitening teeth by applying anoral care product composition comprising a peptide-based whitener to theteeth for a sufficient time to allow the peptide-based whitener to bindto the teeth. The composition may then be rinsed from the teeth. Theoral care product composition may be applied to the teeth using anysuitable method including, but not limited to, brushing, rinsing, andusing an applicator strip or dental floss coated with the composition.

The invention also provides a method for freshening breath by applyingto the oral cavity an oral care product comprising a peptide-basedbreath freshener. The oral care product may be applied as a rinse, atoothpaste, a spray, a gum, or a candy.

The above methods of application of the binding reagents to bodysurfaces are characterized by the ability of the regent to bind to asurface in an aqueous environment and to bind rapidly, often within 5 toabout 60 seconds from the time of first application. The reagents of theinvention are multifaceted bio-adhesives with a multiplicity ofapplications but unified in their water fast nature and rapid and tightbinding characteristics.

EXAMPLES

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various uses andconditions.

The meaning of abbreviations used is as follows: “min” means minute(s),“sec” means second(s), “h” means hour(s), “μL” means microliter(s), “mL”means milliliter(s), “L” means liter(s), “nm” means nanometer(s), “mm”means millimeter(s), “cm” means centimeter(s), “μm” means micrometer(s),“mM” means millimolar, “M” means molar, “mmol” means millimole(s),“μmole” means micromole(s), “g” means gram(s), “μg” means microgram(s),“mg” means milligram(s), “g” means the gravitation constant, “rpm” meansrevolutions per minute, “pfu” means plague forming unit, “BSA” meansbovine serum albumin, “ELISA” means enzyme linked immunosorbent assay,“IPTG” means isopropyl β-D-thiogalactopyranoside, “A” means absorbance,“A₄₅₀” means the absorbance measured at a wavelength of 450 nm, “TBS”means Tris-buffered saline, “TBST-X” means Tris-buffered salinecontaining Tween® 20 where “X” is the weight percent of Tween® 20,“Xgal” means 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside, “SEM”means standard error of the mean, “ESCA” means electron spectroscopy forchemical analysis, “eV” means electron volt(s), “TGA” meansthermogravimetric analysis, “GPC” means gel permeation chromatography,“MW” means molecular weight, “M_(W)” means weight-average molecularweight, “vol %” means volume percent, “NMR” means nuclear magneticresonance spectroscopy, and “MALDI mass spectrometry” means matrixassisted, laser desorption ionization mass spectrometry.

General Methods:

Standard recombinant DNA and molecular cloning techniques used in theExamples are well known in the art and are described by Sambrook, J.,Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, byT. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with GeneFusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1984,and by Ausubel, F. M. et al., Current Protocols in Molecular Biology,Greene Publishing Assoc. and Wiley-Interscience, N.Y., 1987.

Materials and methods suitable for the maintenance and growth ofbacterial cultures are also well known in the art. Techniques suitablefor use in the following Examples may be found in Manual of Methods forGeneral Bacteriology, Phillipp Gerhardt, R. G. E. Murray, Ralph N.Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. BriggsPhillips, eds., American Society for Microbiology, Washington, D.C.,1994, or by Thomas D. Brock in Biotechnology: A Textbook of IndustrialMicrobiology, Second Edition, Sinauer Associates, Inc., Sunderland,Mass., 1989. All reagents, restriction enzymes and materials used forthe growth and maintenance of bacterial cells were obtained from AldrichChemicals (Milwaukee, Wis.), BD Diagnostic Systems (Sparks, Md.), LifeTechnologies (Rockville, Md.), or Sigma Chemical Company (St. Louis,Mo.), unless otherwise specified.

Example 1 Selection of Hair-Binding Phage Peptides Using StandardBiopanning

The purpose of this Example was to identify hair-binding phage peptidesthat bind to normal hair and to bleached hair using standard phagedisplay biopanning.

Phage Display Peptide Libraries:

The phage libraries used in the present invention, Ph.D.-12™ PhageDisplay Peptide Library Kit and Ph.D.-7™ Phage Display Library Kit, werepurchased from New England BioLabs (Beverly, Mass.). These kits arebased on a combinatorial library of random peptide 7 or 12-mers fused toa minor coat protein (pIII) of M13 phage. The displayed peptide isexpressed at the N-terminus of pIII, such that after the signal peptideis cleaved, the first residue of the coat protein is the first residueof the displayed peptide. The Ph.D.-7 and Ph.D.-12 libraries consist ofapproximately 2.8×10⁹ and 2.7×10⁹ sequences, respectively. A volume of10 μL contains about 55 copies of each peptide sequence. Each initialround of experiments was carried out using the original library providedby the manufacturer in order to avoid introducing any bias into theresults.

Preparation of Hair Samples:

The samples used as normal hair were 6-inch medium brown human hairsobtained from International Hair Importers and Products (Bellerose,N.Y.). The hairs were placed in 90% isopropanol for 30 min at roomtemperature and then washed 5 times for 10 min each with deionizedwater. The hairs were air-dried overnight at room temperature.

To prepare the bleached hair samples, the medium brown human hairs wereplaced in 6% H₂O₂, which was adjusted to pH 10.2 with ammoniumhydroxide, for 10 min at room temperature and then washed 5 times for 10min each with deionized water. The hairs were air-dried overnight atroom temperature.

The normal and bleached hair samples were cut into 0.5 to 1 cm lengthsand about 5 to 10 mg of the hairs was placed into wells of a custom24-well biopanning apparatus that had a pig skin bottom. An equal numberof the pig skin bottom wells were left empty. The pig skin bottomapparatus was used as a subtractive procedure to remove phage-peptidesthat have an affinity for skin. This apparatus was created by modifyinga dot blot apparatus (obtained from Schleicher & Schuell, Keene, N.H.)to fit the biopanning process. Specifically, the top 96-well block ofthe dot blot apparatus was replaced by a 24-well block. A 4×6 inchtreated pig skin was placed under the 24-well block and panning wellswith a pig skin bottom were formed by tightening the apparatus. The pigskin was purchased from a local supermarket and stored at −80° C. Beforeuse, the skin was placed in deionized water to thaw, and then blotteddry using a paper towel. The surface of the skin was wiped with 90%isopropanol, and then rinsed with deionized water. The 24-well apparatuswas filled with blocking buffer consisting of 1 mg/mL BSA in TBSTcontaining 0.5% Tween® 20 (TBST-0.5%) and incubated for 1 h at 4° C. Thewells and hairs were washed 5 times with TBST-0.5%. One milliliter ofTBST-0.5% containing 1 mg/mL BSA was added to each well. Then, 10 μL ofthe original phage library (2×10¹¹ pfu), either the 12-mer or 7-merlibrary, was added to the pig skin bottom wells that did not contain ahair sample and the phage library was incubated for 15 min at roomtemperature. The unbound phages were then transferred to pig skin bottomwells containing the hair samples and were incubated for 15 min at roomtemperature. The hair samples and the wells were washed 10 times withTBST-0.5%. The hairs were then transferred to clean, plastic bottomwells of a 24-well plate and 1 mL of a non-specific elution bufferconsisting of 1 mg/mL BSA in 0.2 M glycine-HCl, pH 2.2, was added toeach well and incubated for 10 min to elute the bound phages. Then, 160μL of neutralization buffer consisting of 1 M Tris-HCl, pH 9.2, wasadded to each well. The eluted phages from each well were transferred toa new tube for tittering and sequencing.

To titer the bound phages, the eluted phage was diluted with SM buffer(100 mM NaCl, 12.3 mM MgSO₄-7H₂O, 50 mM Tris-HCl, pH 7.5, and 0.01wt/vol % gelatin) to prepare 10-fold serial dilutions of 10¹ to 10⁴. A10 μL aliquot of each dilution was incubated with 200 μL of mid-logphase E. coli ER2738 (New England BioLabs), grown in LB medium for 20min and then mixed with 3 mL of agarose top (LB medium with 5 mM MgCl₂,and 0.7% agarose) at 45° C. This mixture was spread onto a 5-GAL™/LBagar plate (Sigma Chemical Co.) and incubated overnight at 37° C. TheS-Gal™/LB agar blend contained 5 g of tryptone, 2.5 g of yeast extract,5 g of sodium chloride, 6 g of agar, 150 mg of3,4-cyclohexenoesculetin-β-D-galactopyranoside (S-GAL™), 250 mg offerric ammonium citrate and 15 mg of isopropyl β-D-thiogalactoside(IPTG) in 500 mL of distilled water. The plates were prepared byautoclaving the 5-GAL™/LB for 15 to 20 min at 121-124° C. The singleblack plaques were randomly picked for DNA isolation and sequenceanalysis.

The remaining eluted phages were amplified by incubating with diluted E.coli ER2738, from an overnight culture diluted 1:100 in LB medium, at37° C. for 4.5 h. After this time, the cell culture was centrifuged for30 s and the upper 80% of the supernatant was transferred to a freshtube, ⅙ volume of PEG/NaCl (20% polyethylene glyco-800, 2.5 M sodiumchloride) was added, and the phage was allowed to precipitate overnightat 4° C. The precipitate was collected by centrifugation at 10,000×g at4° C. and the resulting pellet was resuspended in 1 mL of TBS. This wasthe first round of amplified stock. The amplified first round phagestock was then titered according to the same method as described above.For the next round of biopanning, more than 2×10¹¹ pfu of phage stockfrom the first round was used. The biopanning process was repeated for 3to 6 rounds depending on the experiments.

The single plaque lysates were prepared following the manufacture'sinstructions (New England Biolabs) and the single stranded phage genomicDNA was purified using the QIAprep Spin M13 Kit (Qiagen, Valencia,Calif.) and sequenced at the DuPont Sequencing Facility using −96 gIIIsequencing primer (5′-CCCTCATAGTTAGCGTAACG-3′), given as SEQ ID NO:62.The displayed peptide is located immediately after the signal peptide ofgene III.

The amino acid sequences of the eluted normal hair-binding phagepeptides from the 12-mer library isolated from the fifth round ofbiopanning are given in Table 1. The amino acid sequences of the elutedbleached hair-binding phage peptides from the 12-mer library isolatedfrom the fifth round of biopanning are given in Table 2. Repeated aminoacid sequences of the eluted normal hair-binding phage peptides from the7-mer library from 95 randomly selected clones, isolated from the thirdround of biopanning, are given in Table 3.

TABLE 1 Amino Acid Sequences of Eluted Normal Hair-Binding Phage Peptides from  12-Mer Library SEQ Clone Amino Acid IDID Sequence NO: Frequency¹ 1 RVPNKTVTVDGA 5 5 2 DRHKSKYSSTKS 6 2 3KNFPQQKEFPLS 7 2 4 QRNSPPAMSRRD 8 2 5 TRKPNMPHGQYL 9 2 6 KPPHLAKLPFTT 101 7 NKRPPTSHRIHA 11 1 8 NLPRYQPPCKPL 12 1 9 RPPWKKPIPPSE 13 1 10RQRPKDHFFSRP 14 1 11 SVPNKXVTVDGX 15 1 12 TTKWRHRAPVSP 16 1 13WLGKNRIKPRAS 17 1 14 SNFKTPLPLTQS 18 1 15 SVSVGMKPSPRP 3 1 ¹Thefrequency represents the number of identical sequences that occurred outof 23 sequenced clones.

TABLE 2 Amino Acid Sequences of Eluted BleachedHair-Binding Phage Peptides from 12-Mer Library SEQ Clone Amino Acid IDID Sequence NO: Frequency¹ 1 KELQTRNVVQRE 19 8 2 QRNSPPAMSRRD 8 5 3TPTANQFTQSVP 20 2 4 AAGLSQKHERNR 21 2 5 ETVHQTPLSDRP 22 1 6 KNFPQQKEFPLS7 1 7 LPALHIQRHPRM 23 1 8 QPSHSQSHNLRS 24 1 9 RGSQKSKPPRPP 25 1 10THTQKTPLLYYH 26 1 11 TKGSSQAILKST 27 1 ¹The frequency represents thenumber of identical sequences that occurred out of 24 sequenced clones.

TABLE 3 Amino Acid Sequences of Eluted NormalHair-Binding Phage Peptides from 7-Mer Library Clone SEQ ID IDAmino Acid Sequence NO: A DLHTVYH 28 B HIKPPTR 29 D HPVWPAI 30 E MPLYYLQ31 F¹ HLTVPWRGGGSAVPFYSHSQITLPNH 32 G¹ GPHDTSSGGVRPNLHHTSKKEKREN 33RKVPFYSHSVTSRGNV H KHPTYRQ 34 I HPMSAPR 35 J MPKYYLQ 36 ¹There was amultiple DNA fragment insertion in these clones.

Example 2 Selection of High Affinity Hair-Binding Phage Peptides Using aModified Method

The purpose of this Example was to identify hair-binding phage peptideswith a higher binding affinity.

The hairs that were treated with the acidic elution buffer, as describedin Example 1, were washed three more times with the elution buffer andthen washed three times with TBST-0.5%. These hairs, which had acidresistant phage peptides still attached, were used to directly infect500 μL of mid-log phase bacterial host cells, E. coli ER2738 (NewEngland BioLabs), which were then grown in LB medium for 20 min and thenmixed with 3 mL of agarose top (LB medium with 5 mM MgCl₂, and 0.7%agarose) at 45° C. This mixture was spread onto a LB medium/IPTG/S-Gal™plate (LB medium with 15 g/L agar, 0.05 g/L IPTG, and 0.04 g/L S-Gal™)and incubated overnight at 37° C. The black plaques were counted tocalculate the phage titer. The single black plaques were randomly pickedfor DNA isolation and sequencing analysis, as described in Example 1.This process was performed on the normal and bleached hair samples thatwere screened with the 7-mer and 12-mer phage display libraries, asdescribed in Example 1. The amino acid sequences of these high affinity,hair-binding phage peptides are given in Tables 4-7.

TABLE 4 Amino Acid Sequences of High Affinity,Normal Hair-Binding Phage Peptides  from 7-Mer Library Clone Amino AcidSEQ ID ID Sequence NO: D5 GPHDTSSGGVRPNL 33 HHTSKKEKRENRKVPFYSHSVTSRGNV¹ A36 MHAHSIA 37 B41 TAATTSP 38 ¹There was a multiple DNAfragment insertion in this clone.

TABLE 5 Amino Acid Sequencesof High Affinity,Bleached Hair-Binding Phage Peptides from 7-Mer Library Clone Amino AcidSEQ ID ID Sequence NO: D39 LGIPQNL 39 B1 TAATTSP 38

TABLE 6 Amino Acid Sequences of High Affinity,Normal Hair-Binding Phage Peptides from 12-Mer Library Clone Amino AcidSEQ ID ID Sequence NO: C2 AKPISQHLQRGS 40 A3 APPTPAAASATT 41 F9DPTEGARRTIMT 42 A19 EQISGSLVAAPW 43 F4 LDTSFPPVPFHA 44 F35 LPRIANTWSPS45 D21 RTNAADHPAAVT 46 C10 SLNWVTIPGPKI 47 C5 TDMQAPTKSYSN 48 D20TIMTKSPSLSCG 49 C18 TPALDGLRQPLR 50 A20 TYPASRLPLLAP 51 C13 AKTHKHPAPSYS52 G-D20 YPSFSPTYRPAF 53 A23 TDPTPFSISPER 54 F67 SQNWQDSTSYSN 55 F91WHDKPQNSSKST 56 G-F1 LDVESYKGTSMP  4

TABLE 7 Amino Acid Sequences of High Affinity,Bleached Hair-Binding Phage Peptides from 12-Mer Library CloneAmino Acid SEQ ID ID Sequence NO: A5 EQISGSLVAAPW 43 C4 NEVPARNAPWLV 57D30 NSPGYQADSVAIG 58 C44 AKPISQHLQRGS 40 E66 LDTSFPPVPFHA 44 C45SLNWVTIPGPKI 47 E18 TQDSAQKSPSPL 59

Example 3 Selection of High Affinity Fingernail-Binding Phage Peptides

The purpose of this Example was to identify phage peptides that have ahigh binding affinity to fingernails. The modified biopanning methoddescribed in Example 2 was used to identify high affinity,fingernail-binding phage-peptide clones.

Human fingernails were collected from test subjects. The fingernailswere cleaned by brushing with soap solution, rinsed with deionizedwater, and allowed to air-dry at room temperature. The fingernails werethen powdered under liquid N₂, and 10 mg of the fingernails was added toeach well of a 96-well filter plate. The fingernail samples were treatedfor 1 h with blocking buffer consisting of 1 mg/mL BSA in TBST-0.5%, andthen washed with TBST-0.5%. The fingernail samples were incubated withphage library (Ph.D-12 Phage Display Peptide Library Kit), and washed 10times using the same conditions described in Example 1. After the acidicelution step, described in Example 1, the fingernail samples were washedthree more times with the elution buffer and then washed three timeswith TBST-0.5%. The acid-treated fingernails, which had acid resistantphage peptides still attached, were used to directly infect E. coliER2738 cells as described in Example 2. This biopanning process wasrepeated three times. A total of 75 single black phage plaques werepicked randomly for DNA isolation and sequencing analysis and tworepeated clones were identified. The amino acid sequences of these phagepeptides are listed in Table 8. These fingernail binding peptides werealso found to bind well to bleached hair.

TABLE 8 Amino Acid Sequences of High AffinityFingernail-Binding Phage Peptides Clone Amino Acid SEQ ID ID SequenceNO: Frequency¹ F01 ALPRIANTWSPS 60 15 D05 YPSFSPTYRPAF 53 26 ¹Thefrequency represents the number of identical sequences that occurred outof 75 sequenced clones.

Example 4 Selection of High Affinity Skin-Binding Phage Peptides

The purpose of this Example was to identify phage peptides that have ahigh binding affinity to skin. The modified biopanning method describedin Examples 2 and 4 was used to identify the high affinity, skin-bindingphage-peptide clones. Pig skin served as a model for human skin in theprocess.

The pig skin was prepared as described in Example 1. Three rounds ofscreenings were performed with the custom, pig skin bottom biopanningapparatus using the same procedure described in Example 4. A total of 28single black phage plaques were picked randomly for DNA isolation andsequencing analysis and one repeated clone was identified. The aminoacid sequence of this phage peptide, which appeared 9 times out of the28 sequences, was TPFHSPENAPGS, given as SEQ ID NO:61.

Example 5 Quantitative Characterization of the Binding Affinity ofHair-Binding Phage Clones

The purpose of this Example was to quantify the binding affinity ofphage clones by titering and ELISA.

Titering of Hair-Binding Phage Clones:

Phage clones displaying specific peptides were used for comparing thebinding characteristics of different peptide sequences. A titer-basedassay was used to quantify the phage binding. This assay measures theoutput pfu retained by 10 mg of hair surfaces, having a signal to noiseratio of 10³ to 10⁴. The input for all the phage clones was 10¹⁴ pfu. Itshould be emphasized that this assay measures the peptide-expressingphage particle, rather than peptide binding.

Normal hairs were cut into 0.5 cm lengths and 10 mg of the cut hair wasplaced in each well of a 96-well filter plate (Qiagen). Then, the wellswere filled with blocking buffer containing 1 mg/mL BSA in TBST-0.5% andincubated for 1 h at 4° C. The hairs were washed 5 times with TBST-0.5%.The wells were then filled with 1 mL of TBST-0.5% containing 1 mg/mL BSAand then purified phage clones (10¹⁴ pfu) were added to each well. Thehair samples were incubated for 15 min at room temperature and thenwashed 10 times with TBST-0.5%. The hairs were transferred to a cleanwell and 1.0 mL of a non-specific elution buffer, consisting of 1 mg/mLBSA in 0.2 M Glycine-HCl at pH 2.2, was added to each well. The sampleswere incubated for 10 min and then 160 μL of neutralization buffer (1 MTris-HCl, pH 9.2) was added to each well. The eluted phages from eachwell were transferred to a new tube for titering and sequencinganalysis.

To titer the bound phages, the eluted phage was diluted with SM bufferto prepare 10-fold serial dilutions of 10¹ to 10⁸. A 10 μL aliquot ofeach dilution was incubated with 200 μL of mid-log phase E. coli ER2738(New England BioLabs), and grown in LB medium for 20 min and then mixedwith 3 mL of agarose top (LB medium with 5 mM MgCl₂, and 0.7% agarose)at 45° C. This mixture was spread onto a LB medium/IPTG/Xgal plate (LBmedium with 15 g/L agar, 0.05 g/L IPTG, and 0.04 g/L Xgal) and incubatedovernight at 37° C. The blue plaques were counted to calculate the phagetiters, which are given in Table 9.

TABLE 9 Titer of Hair-Binding Phage Clones Clone ID SEQ ID NO: PhageTiter A 28 7.50 × 10⁴ B 29 1.21 × 10⁵ D 30 8.20 × 10⁴ E 31 1.70 × 10⁵ F32 1.11 × 10⁶ G 33 1.67 × 10⁸ H 34 1.30 × 10⁶ 1 35 1.17 × 10⁶ J 36 1.24× 10⁶

Characterization of Hair-Binding Phage Clones by ELISA:

Enzyme-linked immunosorbent assay (ELISA) was used to evaluate thehair-binding specificity of selected phage-peptide clones. Phage-peptideclones identified in Examples 1 and 2 along with a randomly chosencontrol G-F9, KHGPDLLRSAPR (given as SEQ ID NO:63) were amplified. Morethan 10¹⁴ pfu phages were added to pre-blocked hair surfaces. The sameamount of phages was also added to pre-blocked pig skin surfaces as acontrol to demonstrate the hair-binding specificity.

A unique hair or pig skin-bottom 96-well apparatus was created byapplying one layer of Parafilm® under the top 96-well block of aMinifold I Dot-Blot System (Schleicher & Schuell, Inc., Keene, N.H.),adding hair or a layer of hairless pig skin on top of the Parafilm®cover, and then tightening the apparatus. For each clone to be tested,the hair-covered well was incubated for 1 h at room temperature with 200μL of blocking buffer, consisting of 2% non-fat dry milk (Schleicher &Schuell, Inc.) in TBS. A second Minifold system with pig skin at thebottom of the wells was treated with blocking buffer simultaneously toserve as a control. The blocking buffer was removed by inverting thesystems and blotting them dry with paper towels. The systems were rinsed6 times with wash buffer consisting of TBST-0.05%. The wells were filledwith 200 μL of TBST-0.5% containing 1 mg/mL BSA and then 10 μL (over10¹² copies) of purified phage stock was added to each well. The sampleswere incubated at 37° C. for 15 min with slow shaking. The non-bindingphage was removed by washing the wells 10 to 20 times with TBST-0.5%.Then, 100 μL of horseradish peroxidase/anti-M13 antibody conjugate(Amersham USA, Piscataway, N.J.), diluted 1:500 in the blocking buffer,was added to each well and incubated for 1 h at room temperature. Theconjugate solution was removed and the wells were washed 6 times withTBST-0.05%. TMB substrate (200 μL), obtained from Pierce Biotechnology(Rockford, Ill.) was added to each well and the color was allowed todevelop for between 5 to 30 min, typically for 10 min, at roomtemperature. Then, stop solution (200 μL of 2 M H₂SO₄) was added to eachwell and the solution was transferred to a 96-well plate and the A₄₅₀was measured using a microplate spectrophotometer (Molecular Devices,Sunnyvale, Calif.). The resulting absorbance values, reported as themean of at least three replicates, and the standard error of the mean(SEM) are given in Table 10.

TABLE 10 Results of ELISA Assay with Skin and Hair SEQ ID Hair Pig SkinClone ID NO: A₄₅₀ SEM A₄₅₀ SEM G-F9 63 0.074 0.057 −0.137 0.015(Control) D21 46 1.051 0.16 0.04 0.021 D39 39 0.685 0.136 0.086 0.019 D533 0.652 0.222 0.104 0.023 A36 37 0.585 0.222 0.173 0.029 C5 48 0.5480.263 0.047 0.037 C10 47 0.542 0.105 0.032 0.012 A5 43 0.431 0.107 0.2560.022 B1 38 0.42 0.152 0.127 0.023 D30 58 0.414 0.119 0.287 0.045 C13 520.375 0.117 0.024 0.016 C18 50 0.34 0.197 0.132 0.023

As can be seen from the data in Table 10, all the hair-binding cloneshad a significantly higher binding affinity for hair than the control.Moreover, the hair-binding clones exhibited various degrees ofselectivity for hair compared to pig skin. Clone D21 had the highestselectivity for hair, having a very strong affinity for hair and a verylow affinity for pig skin.

Example 6 Confirmation of Peptide Binding Specificity and Affinity

The purpose of this Example was to test the peptide binding sitespecificity and affinity of the hair-binding peptide D21 using acompetition ELISA. The ELISA assay only detects phage particles thatremain bound to the hair surface. Therefore, if the synthetic peptidecompetes with the phage particle for the same binding site on hairsurface, the addition of the synthetic peptide into the ELISA systemwill significantly reduce the ELISA results due to the peptidecompetition.

The synthetic hair-binding peptide D21, given as SEQ ID NO:46, wassynthesized by SynPep (Dublin, Calif.). As a control, an unrelatedsynthetic skin-binding peptide, given as SEQ ID NO:61, was added to thesystem. The experimental conditions were similar to those used in theELISA method described in Example 5. Briefly, 100 μL of Binding Buffer(1×TBS with 0.1% Tween®20 and 1 mg/mL BSA) and 10¹¹ pfu of the pure D21phage particles were added to each well of the 96-well filter plate,which contained a sample of normal hair. The synthetic peptide (100 μg)was added to each well (corresponding to concentration of 0.8 mM). Thereactions were carried out at room temperature for 1 h with gentleshaking, followed by five washes with TBST-0.5%. The remaining stepswere identical to the those used in the ELISA method described inExample 5. The ELISA results, presented as the absorbance at 450 nm(A₄₅₀), are shown in Table 11. Each individual ELISA test was performedin triplicate; the values in Table 11 are the means of the triplicatedeterminations.

TABLE 11 Results of Peptide Competition ELISA Sample A₄₅₀ SEMAntibody-Conjugate 0.199 0.031 Phage D21 1.878 0.104 Phage D21 and D211.022 0.204 Peptide Phage D21 and 2.141 0.083 Control Peptide

These results demonstrated that the synthetic peptide D21 does competewith the phage clone D21 for the same binding sites on the hair surface.

Example 7 Selection of Shampoo-Resistant Hair-Binding Phage-PeptidesUsing Biopanning

The purpose of this Example was to select shampoo-resistant hair-bindingphage-peptides using biopanning with shampoo washes.

In order to select shampoo-resistant hair-binding peptides, a biopanningexperiment using 12-mer phage peptide libraries against normal andbleached hairs was performed, as described in Example 2. Instead ofusing normal TBST buffer to wash-off the unbounded phages, thephage-complexed hairs were washed with 10%, 30% and 50% shampoosolutions (Pantene Pro-V shampoo, Sheer Volume, Proctor & Gamble,Cincinnati, Ohio), for 5 min in separate tubes, followed by six TBSbuffer washes. The washed hairs were directly used to infect hostbacterial cells as described in the modified biopanning method,described in Example 2.

A potential problem with this method is the effect of the shampoo on thephage's ability to infect bacterial host cells. In a control experiment,a known amount of phage particles was added to a 10% shampoo solutionfor 5 min, and then a portion of the solution was used to infectbacterial cells. The titer of the shampoo-treated phage was 90% lowerthan that of the untreated phage. The 30% and 50% shampoo treatmentsgave even more severe damage to the phage's ability to infect hostcells. Nevertheless, two shampoo-resistant hair-binding phage-peptideswere identified, as shown in Table 12.

TABLE 12 Peptide Sequences of Shampoo-ResistantHair-binding Phage Peptides Identified Using the Biopanning MethodSEQ ID Clone Sequence Target NO: I-B5 TPPELLHGDPRS Normal and 66Bleached Hair H-B1 TPPTNVLMLATK Normal Hair 69

Example 8 Selection of Shampoo-Resistant Hair-Binding Phage-PeptidesUsing PCR

The purpose of this Example was to select shampoo-resistant hair-bindingphage-peptides using a PCR method to avoid the problem of shampooinduced damage to the phage. This principle of the PCR method is thatDNA fragments inside the phage particle can be recovered using PCR,regardless of the phage's viability, and that the recovered DNAfragments, corresponding to the hair-binding peptide sequences, can thenbeen cloned back into a phage vector and packaged into healthy phageparticles.

Biopanning experiments were performed using 7-mer and 12-merphage-peptide libraries against normal and bleached hairs, as describedin Example 1. After the final wash, the phage-treated hairs weresubjected to 5 min of shampoo washes, followed by six TBS buffer washes.The shampoo-washed hairs were put into a new tube filled with 1 mL ofwater, and boiled for 15 min to release the DNA. This DNA-containing,boiled solution was used as a DNA template for PCR reactions. Theprimers used in the PCR reaction were primers: M13KE-1412 Forward5′-CAAGCCTCAGCGACCGAATA-3′, given as SEQ ID NO:67 and M13KE-1794 Reverse5′-CGTAACACTGAGTTTCGTCACCA-3′, given SEQ ID NO:68. The PCR conditionswere: 3 min denaturing at 96° C., followed by 35 cycles of 94° C. for 30sec, 50° C. for 30 sec and 60° C. for 2 min. The PCR products (˜400 bp),and M13KE vector (New England BioLabs) were digested with restrictionenzymes Eag I and Acc65 I. The ligation and transformation conditions,as described in the Ph.D.™ Peptide Display Cloning System (New EnglandBiolabs), were used. The amino acid sequence of the resultingshampoo-resistant hair-binding phage-peptide is NTSQLST, given as SEQ IDNO:70.

Example 9 Determination of the Affinity of Hair-Binding and Skin-BindingPeptides

The purpose of this Example was to determine the affinity of thehair-binding and skin-binding peptides for their respective substrates,measured as MB₅₀ values, using an ELISA assay.

Hair-binding and skin-binding peptides were synthesized by Syn Pep Inc.(Dublin, Calif.). The peptides were biotinylated by adding abiotinylated lysine residue at the C-terminus of the amino acid bindingsequences for detection purposes and an amidated cysteine was added tothe C-terminus of the sequence. The amino acid sequences of the peptidestested are given as SEQ ID NOs:71-74, as shown in Table 13.

For hair samples, the procedure used was as follows. The setup of thesurface specific 96-well system used was the same as that described inExample 5. Briefly, the 96-wells with hair or pig skin surfaces wereblocked with blocking buffer (SUPERBLOCK™ from Pierce Chemical Co.,Rockford, Ill.) at room temperature for 1 h, followed by six washes withTBST-0.5%, 2 min each, at room temperature. Various concentrations ofbiotinylated, binding peptide were added to each well, incubated for 15min at 37° C., and washed six times with TBST-0.5%, 2 min each, at roomtemperature. Then, streptavidin-horseradish peroxidase (HRP) conjugate(Pierce Chemical Co.) was added to each well (1.0 μg per well), andincubated for 1 h at room temperature. After the incubation, the wellswere washed six times with TBST-0.5%, 2 min each at room temperature.Finally, the color development and the measurement were performed asdescribed in Example 5.

For the measurement of MB₅₀ of the peptide-skin complexes, the followingprocedure was used. First, the pigskin was treated to block theendogenous biotin in the skin. This was done by adding streptavidin tothe blocking buffer. After blocking the pigskin sample, the skin wastreated with D-biotin to block the excess streptavidin binding sites.The remaining steps were identical to those used for the hair samples.

The results were plotted as A₄₅₀ versus the concentration of peptideusing GraphPad Prism 4.0 (GraphPad Software, Inc., San Diego, Calif.).The MB₅₀ values were calculated from Scatchard plots and are summarizedin Table 13. The results demonstrate that the binding affinity of thehair-binding peptides (D21, F35, and I-B5) and the skin binding peptide(SEQ ID NO:61) for their respective substrate was high, while thebinding affinity of the hair-binding peptides (D-21 and I-B5) for skinwas relatively low.

TABLE 13 Summary of MB₅₀ Values for Hair and Skin-Binding PeptidesPeptide Sequence Binding Peptide Tested* Substrate MB₅₀, M D21 SEQ IDNO: 71 Normal Hair 2 × 10⁻⁶ F35 SEQ ID NO: 72 Bleached Hair 3 × 10⁻⁶I-B5 SEQ ID NO: 73 Normal and 3 × 10⁻⁷ Bleached Hair D21 SEQ ID NO: 71Pig Skin 4 × 10⁻⁵ I-B5 SEQ ID NO: 73 Pig Skin >1 × 10⁻⁴   SEQ ID NO: 61SEQ ID NO: 74 Pig Skin 7 × 10⁻⁷ *The peptides tested were biotinylatedat the C-terminus of the amino acid binding sequences and an amidatedcysteine was added to the C-terminus of the binding sequence.

Example 10 Preparation of a Peptide-Based-Carbon Black Hair Colorant

The purpose of this Example was to prepare a peptide-based-carbon blackhair colorant by covalently linking the hair-binding peptide D21, givenas SEQ ID NO:46, to the surface of carbon black particles. The surfaceof the carbon black particles was functionalized by reaction with2,2′-azobis(2methylpropionamide)-dihydrochloride to introduce free aminogroups. The functionalized carbon black particles were then covalentlylinked to the specific hair-binding peptide.

Functionalization of Carbon Black Surface:

Carbon black (NIPEX® 160-IQ from Degussa, Allendale, N.J.), 2.0 g, and1.0 g of 2,2′-Azobis(2-methylpropionamide)dihydrochloride (Aldrich,Milwaukee, Wis.) were added to a 100 mL round-bottom flask and 30 mL ofdioxane was added. The flask was purged with nitrogen for 5 min. Then,the flask was sealed with a rubber septum and the reaction mixture wasstirred at 65° C. for 14 h. After this time, 50 mL of deionized water,prepared with a Nanopure water purification system(Barnstead/Thermolyne, Dubuque, Iowa), was added to the mixture. Thediluted solution was centrifuged to collect the functionalized carbonblack particles and to remove the organic solvent and unreactedreagents. The carbon black particles were washed with deionized waterand centrifuged. This washing and centrifuging process was repeated 2more times. The functionalized carbon black particles were then dried bylyophilization.

Synthesis of t-Boc-Protected Hair-Binding Peptide from Phage Clone D21

The purpose of this reaction was to protect the amino end group of thehair-binding peptide. The hair-binding peptide from phage clone D21(0.25 g), given as SEQ ID NO:46 (95% purity, obtained from SynPep,Dublin, Calif.) was mixed with 2.5 mL of deionized water in a 25 mLround-bottom flask. Then, 20 mg of NaOH and 0.25 mL of t-butyl alcoholwere added. After stirring the mixture for 2 min, 0.12 g ofdi-tert-butyl dicarbonate (t-Boc anhydride) (Aldrich) was addeddropwise. The flask was sealed with a rubber septum and the reactionmixture was stirred overnight at room temperature. The reaction mixturewas clear at the beginning of the reaction and became cloudy and then,precipitated after 1 h. Upon addition of water (10 mL), the reactionmixture formed a milky emulsion, which was then extracted three timeswith 5 mL portions of methylene chloride. The organic layer was washedtwice with 5 mL portions of deionized water. The clear water layers wereall combined and dried by lyophilization, yielding 0.20 g of a fluffywhite powder (80% yield). The product was analyzed by liquidchromatography-mass spectrometry (LC-MS) and was found to have amolecular weight of 1323 g/mol, with a purity of 90% by weight.

Coupling of Amino-Functionalized Carbon Black with t-Boc-D21-Peptide:

Amino-functionalized carbon black (87 mg), t-Boc-D21-peptide (80 mg) anddicyclohexyl carbodiimide (22 mg) were added to 3 mL of tetrahydrofuran(THF). A solution of dimethyl aminopyridine (17 μL) in several drops ofTHF was added dropwise to this mixture with stirring. The resulting darksuspension was heated to 40° C. for 6 h with stirring, followed bystirring overnight at room temperature. Trifluoroacetic acid (0.6 mL)was added to the product and the mixture was stirred for another 6 h.Then, 5 mL of deionized water was added to the reaction mixture. Themixture was centrifuged at 3,500 rpm for 2 min and the supernatant wasdecanted. The solid remaining in the centrifuge tube was washed withdeionized water and centrifuged again. This washing was repeated untilthe pH of supernatant reached approximately 6.0. The dark residue wasthen dried using a lyophilizer for 2 days, yielding a dark powder.

Example 11 Hair Dyeing Using a Peptide-Based-Carbon Black Hair Colorant

The purpose of this Example was to dye a sample of natural white hairusing the peptide-based-carbon black hair colorant prepared in Example10.

A bundle of natural white hair (approximately 100 pieces) (fromInternational Hair Importers and Products Inc., Bellerose, N.Y.) wascleaned by mixing with 10 mL of 50% isopropanol for 30 min and thenwashed at least 5 times with distilled water. After drying in air, thecleaned hair was immersed for 30 min in a solution containing 50 mg ofthe hair-binding D21 peptide-carbon black hair colorant, described inExample 10, dissolved in 10 mL of distilled water. After dying, the hairwas washed at least 5 times with distilled water. The original naturalwhite hair became light black. The dyed hair was washed three times witha 30% shampoo solution (Pantene Pro-V shampoo) by immersing the hair inthe shampoo solution and stirring with a glass pipette. The hair wasthen rinsed at least 10 times with distilled water. The final color ofthe dyed, natural white hair was very light black.

Example 12 Preparation of a Peptide-Based Hair Conditioner

The purpose of this Example was to prepare a peptide-based hairconditioner by covalently linking the hair-binding D21 peptide, given asSEQ ID NO:46, with behenyl alcohol using carbodiimide coupling.

Behenyl alcohol (Aldrich), 81.7 mg, and 62.0 mg of dicyclohexylcarbodiimide (DCC) were dissolved in 2.0 mL of THF in a 25 mLround-bottom flask. A solution containing 0.25 g of the9-fluorenylmethyloxycarbonyl (Fmoc) N-terminal protected form of SEQ IDNO:46 (95% purity, obtained from Syn Pep, Dublin, Calif.) in 2.0 mLdimethylformamide (DMF) was added to the above mixture. Then, 50 μL ofdimethylaminopyridine (DMAP) was added to the reaction mixture. Withstirring, the reaction mixture was maintained at 40° C. for 3 h, andthen at room temperature overnight. Then the solvent was evaporatedunder vacuum at room temperature for 4 h. After this time, the mixturewas dissolved in 25 mL of ethyl acetate, and the unreacted peptide wasextracted 3 times with water using 10 mL of deionized water for eachextraction. The ethyl acetate phase was isolated and the ethyl acetatewas removed using a rotary evaporator. The resulting solid product wasdissolved in a solvent consisting of 2.5 mL of THF and 2.5 mL of DMF,and 1.5 mL of piperidine was added to deblock the amino group of the D21peptide. This mixture was stirred for 2 h at room temperature and thenthe solvents were removed by rotary evaporation under vacuum. The finalproduct was characterized by LC/MS.

Example 13 Preparation of a Peptide-Based Hair Conditioner

The purpose of this Example was to prepare a peptide-based hairconditioner by covalently linking the hair-binding, cysteine-attachedD21 peptide, given as SEQ ID NO:64, with octylamine using theheterobifunctional cross-linking agent 3-maleimidopropionic acidN-hydroxysuccinimide ester.

Octylamine, obtained from Aldrich (Milwaukee, Wis.) was diluted byadding 11.6 mg to 0.3 mL of DMF. This diluted solution was added to astirred solution containing 25 mg of 3-maleimidopropionic acidN-hydroxysuccinimide ester (Aldrich) and 5 mg of diisopropylethylamine(Aldrich) in 0.2 mL of DMF in a 5 mL round bottom flask. The reactionmixture turned turbid immediately and then became clear several minuteslater. The solution was stirred for another 4 h. The solution was thendried under high vacuum. The product, octylamine-attachedmaleimidopropionate, was purified by column chromatography using aSilica gel 60 (EMD Chemicals, formerly EM Science, Gibbstown, N.J.)column and DMF/ether as the eluent.

Approximately 12 mg of the above product was placed into a 5 mL roundbottom flask and 50 mg of cysteine-attached D21 peptide (obtained fromSynPep, Dublin, Calif.), given as SEQ ID NO:64, and 0.5 mL of 0.1 Mphosphate buffer at pH 7.2 were added. The cysteine-attached D21 peptidehas 3 glycine residues and a cysteine attached to the end of the peptidebinding sequence of the hair-binding D21 peptide (SEQ ID NO:46). Thismixture was stirred at room temperature for 6 h. The final product, theC8-D21 peptide hair conditioner, was purified by extraction withwater/ether.

Example 14 Preparation of a Peptide-Based-Carbon Black Hair Colorant

The purpose of this Example was to prepare a peptide-based carbon blackhair colorant using carbon black that was functionalized with ethanolamine. The number of peptides attached to the carbon black surface wasestimated from chemical analyses.

Preparation of Acid Functionalized Carbon Black Particles:

In a 1,000 mL beaker was added 25.5 g of carbon black (Nipex-160-IQ fromDegussa, 100 g of ammonium persulfate [(NH₄)₂S₂O₈] (98% from Aldrich),and 333 mL of 1.0 M H₂SO₄ (98%, GR grade from EMD Chemicals) aqueoussolution. The mixture was stirred with a magnetic stir plate for 24 h atroom temperature. After this time, the reaction mixture was transferredto a 500 mL plastic centrifuge tube and centrifuged at 8,500 rpm for 20min. The supernatant became clear and was removed. The product waswashed 6 times with deionized water using centrifugation to collect theproduct after each wash. The final product was neutral (pH=6.0) and wasdried by lyophilization for 24 h. The average size of the functionalizedcarbon black particles was 100 nm, as measured using a particle sizeanalyzer (Microtrac Ultrafine Particle Analyzer, Microtrac Inc.,Montgomeryville, Pa.).

Preparation of Amino-Functionalized Carbon Black Using Ethanolamine:

Two grams of the dried, acid functionalized carbon black, 25 mL ofethanolamine (99% from Aldrich) and 1 mL of concentrated H₂SO₄ (98%, GRgrade from EMD Chemicals) were mixed in a 100 mL round bottom flask. Themixture was stirred rapidly with a magnetic stirrer and refluxed for 6h. After the mixture cooled to room temperature, a sufficient amount ofammonium hydroxide (28.0-30.0% of NH₃ from EMD Chemicals) was added toneutralize the mixture. Then, the mixture was centrifuged and washedwith water, as described in Example 6. The final product was neutral(pH=6.0) and was dried by lyophilization for 24 h. The dried, aminofunctionalized carbon black was readily dispersed in water.

The surface composition of the functionalized carbon black was analyzedby ESCA at the DuPont Corporate Center for Analytical Science. In ESCA,monoenergetic X-rays are focused onto the surface of a material toexcite surface atoms. Core and valence shell electrons with energiescharacteristic of elements in the top 10 nm of the surface are ejectedand their energy analyzed to obtain qualitative and quantitativeinformation on surface composition. The kinetic energy of the electronsemitted provides information about the functional groups and oxidationstates of the surface species. In this Example, the X-ray source usedwas a magnesium anode with an energy of 1253.6 eV. The samples wereanalyzed at a 45 degree exit angle (approximately 5 to 10 nm samplingdepth). The ESCA analysis results are shown in Table 14. Forethanolamine-functionalized carbon black, the surface was mainlycomposed of unreacted —COOH groups and —C(═O)—OCH₂CH₂NH₂ groups. Tocalculate the ratio of amine (y) to carboxylic acid groups (x), a simpleequation was used, specifically, y/(x+y)=(N %/14)/(O %/32) forethanolamine. The results are given in Table 14.

TABLE 14 Results of ESCA Analysis of Functionalized Carbon Black SampleC % O % N % S % —NH₂/—COOH Acid Functionalized 89 10 ND* 0.1 0 CarbonBlack Ethanolamine 87 10 2.6 ND 1.47 Functionalized Carbon Black *NDmeans not detectableCoupling of Amino-Functionalized Carbon Black with t-Boc-D21-Peptide:

The amino-functionalized carbon black particles were then covalentlylinked to the specific hair-binding peptide D21, given as SEQ ID NO:46.The t-Boc protected D21 peptide was synthesized as described in Example10. Then, amino-functionalized carbon black (87 mg), t-Boc-D21-peptide(80 mg) and dicyclohexyl carbodiimide (DCC) (22 mg) were added to 3 mLof tetrahydrofuran (THF). A solution of dimethylaminopyridine (DMAP) (17μL) in several drops of THF was added dropwise to this mixture withstirring. The resulting dark suspension was heated to 40° C. for 6 hwith stirring, followed by stirring overnight at room temperature. Toremove the t-Boc protecting group from the D21 peptide, trifluoroaceticacid (TFA) (0.6 mL) was added to the product and the mixture was stirredfor another 6 h. Then, 5 mL of deionized water was added to the reactionmixture. The mixture was centrifuged at 3,500 rpm for 2 min and thesupernatant was decanted. The solid remaining in the centrifuge tube waswashed with deionized water and centrifuged again. This washing wasrepeated until the pH of supernatant reached approximately 6.0. The darkresidue was then dried using a lyophilizer for 2 days, yielding a darkpowder.

The amino-functionalized carbon black particles and the peptide-linkedcarbon black particles were analyzed by ESCA, elemental analysis, andTGA (thermogravimetric analysis). The analytical results showed that theorganic layer on the carbon black modified with ethanolamine wasapproximately 12% of the total weight. After the D21 peptides wereattached to the carbon black particles, the peptide weight percentagewas in the range of 18-30%. Therefore, for a 100 nm carbon blackparticle, a total of 9.5×10⁴ molecules were attached to the surfaceafter reacting with ethanolamine, and a total of 7,700 D21 peptidemolecules were attached to the carbon black surface after reaction withthe peptide. A calculation of the peptide density on the carbon blacksurface, revealed that each D21 peptide occupied 4 nm², which iscomparable to the peptide density attached to the phage, approximately12 nm².

Example 15 Specificity of the Peptide-Based-Carbon Black Hair Colorant

The purpose of this Example was to demonstrate the specificity of theD21 peptide-carbon black hair colorant.

The D21 peptide-based-carbon black colorant was prepared as described inExample 14.

A piece of pig skin (10 cm×10 cm), obtained from a local supermarket,was cleaned by mixing with 30 mL of 30% isopropanol for 10 min and thenwashed at least 5 times with distilled water. After drying in air, thecleaned pig skin was immersed in a plate holder with multiple wellscontaining a solution of 50 mg of the D21 peptide-carbon black colorantdissolved in 10 mL of distilled water. After applying the colorant for15 min, the pig skin was washed three times with a 30% shampoo solution(Pantene Pro-V shampoo) by dropping the shampoo solution into the wellsand decanting it. Then, the pig skin was rinsed 5 times with distilledwater.

A normal white hair sample, obtained from International Hair Importersand Products (Bellerose, N.Y.), was treated in the same manner as thepig skin.

After washing, the pig skin showed negligible dark color, while the hairwas very light black. These results demonstrate that the D21peptide-carbon black colorant has specific binding to hair, but not toskin.

Example 16 Preparation of a Peptide-Polysiloxane Hair Conditioner

The purpose of this Example was to synthesize a D21 peptide-polysiloxanehair conditioner. The reactive side functional groups of the D21peptide, given as SEQ ID NO:46, were fully protected so that thereaction with the polysiloxane proceeded only with the C-terminal groupof the peptide. In addition, a tripeptide spacer, consisting of glycineresidues, was added to the C-terminal end of the binding sequence.

Fifty milligrams of the fully protected D21 peptideFmoc-R(Pbf)T(tBu)N(Trt)AAD(OtBu)H(Trt)PAAVT(tBu)GGG (where Fmoc meansfluorenylmethoxylcarbonyl; Pbf means2,2,6,4,7-pentamethyldihydrobenzofuran-5-sulfonyl; tBu means t-butyl;Trt means trityl; and Otbu means t-butoxyl) (MW 2522, 0.02 mmol, 95%purity from SynPep, Dublin, Calif.), given as SEQ ID NO:78 was dissolvedin 1 mL of dimethylformamide (DMF, from E. Merck, Darmstadt, Germany) ina 5 mL round bottom flask. Polysiloxane fluid 2-8566 (77 mg) (N%=0.875%, 0.024 mmol of —NH₂, from Dow Corning, Midland, Mich.) wasdissolved in 2 mL of THF (E. Merck) in a sample vial, then transferredinto the round bottom flask containing the peptide solution. Then, 5 mgof dicyclohexyl carbodiimide (DCC, 0.024 mmol) and 5 μL ofdimethylaminopyridine (DMAP) were added to the flask. The flask wassealed with a rubber stopper and the reaction mixture was stirred at 50°C. for 5 h and then, at room temperature overnight. After the reactionwas completed, the solvent was pumped out under vacuum. After drying,122 mg of the solid product was obtained. The yield was about 90%.

The solid product was dissolved in N,N-dimethylacetamide (DMAC, from EMDChemicals) and 5 mg/mL of the product solution in DMAC was prepared forGPC (gel permeation chromatography) analysis with refractive indexdetection to determine the molecular weight. The original polysiloxane(Dow Corning 2-8566) was not soluble in DMAC and was not observed in theseparation region of the chromatogram. The D21 peptide had a sharp, lowmolecular weight peak, and the product sample contained 2 peaks, onefrom the free D21 peptide and a broad peak, which was attributed topolysiloxane grafted with D21 peptide. The weight-average molecularweight (M_(W)) was calculated from polymethylmethacrylate (PMMA)standards. The M_(W) of D21 peptide and the peptide-polysiloxaneconditioner were 4.7×10³, and 4.4×10⁴, respectively.

A cleavage reagent (referred to as Reagent K) having the followingcomposition:

trifluoroacetic acid/H2O/thioanisole/ethanedithiol/phenol(85:5:5:2.5:2.5, by volume) was used to cleave the protecting groupsfrom the side functional groups of the D21 peptide. Reagent K (1 mL) waspre-cooled to −20° C. and then, added to 100 mg of the D21peptide-polysiloxane conditioner. The mixture was stirred for 3-4 h atroom temperature and then Reagent K was removed under high vacuum. Then,the Fmoc protecting group was removed from the N-terminus of the peptideby adding 61.2 mg of 20 vol % piperidine in DMF to the mixture andstirring for 30 min, followed by pumping under high vacuum. The finalproduct was not completely soluble in THF, DMF, or DMAC. GPC analysis ofthe final product was not possible because of the low solubility.

Example 17 Effectiveness of Peptide-Based Hair Conditioner

The purpose of this Example was to demonstrate the effectiveness of apeptide-based hair conditioner in reducing frictional forces in humanhair fibers and to compare its performance against a commercialconditioning agent. Fiber friction is a significant contributor tocombing behavior of hair fiber assemblies (i.e., multiple fibers). Thesingle hair fiber characterization of frictional forces can be relatedto the combing behavior of the hair assembly. Interfiber frictionstudies illustrate the improvement to the hair surface from conditionerapplications. The lower the interfiber friction, the smoother the hairlooks and feels, and the easier it is to comb. The interfiber frictionmeasurement method employed in this Example is one of a few hair fibertests to give hard, quantitative data and is generally accepted in theindustry.

The peptide-based hair conditioner described in Example 12, whichconsists of the hair-binding peptide given as SEQ ID NO:46 covalentlylinked to behenyl alcohol, was used in a formulation consisting of amixture of 0.25% by weight of the peptide-based conditioner and 1.5% byweight of Performix™ Lecithin (ADM Lecithin, Decatur. IL) in distilledwater. The aqueous solution was mixed at 7000 rpm for 4 min using aSilverson L4RT-A High Shear Laboratory Mixer (Silverson Machines, Inc.,East Longmeadow, Mass.) with a general purpose disintegrating head and a0.95 cm mini-micro tubular frame. A 0.5% solution of Dow Corning® 929Cationic Emulsion (Dow Corning Corp., Midland, Mich.), a commercialconditioning agent, in distilled water was prepared using identicalmixing conditions.

European dark brown hair swatches (International Hair Importers andProducts) were cleaned before testing by immersing in isopropanol for 30min, then washing 10 times with distilled water. Single hair fibers fromthese swatches were sent to Textile Research Institute (TRI), Princeton,N.J., for friction testing. At TRI, the hair fibers were immersed in theconditioner solutions for 5 min at approximately 35° C. withoutagitation. Afterwards, they were rinsed for 1 min in lukewarm water andthen dried overnight at 21° C. and 65% relative humidity.

Frictional force measurements of treated hair fibers were measured bythe Interfiber friction test using a single-fiber friction apparatus, asdescribed by Kamath et al. (J. Appl. Polymer Sci., 85:394-414 (2002)).Hair fibers were evaluated at high normal forces (high load) (0.74 g)against a chromed steel wire, crosshead speed of 1 mm/min, using anInstron Tensile Testing machine. Low normal forces (low load) (8.5 mg)were measured against another single hair fiber using the TRI/Scan™Surface Force Analyzer (Textile Research Institute). This apparatusmeasures small forces with a Cahn® microbalance (mass resolution of 0.1mg) and features a computer controlled stage. The results of thesemeasurements are given in Table 15.

TABLE 15 Results of Friction Measurements Friction Force (F_(f))Cationic Peptide Cationic Peptide Low Emulsion Conditioner High EmulsionConditioner Load F_(f) (mg) F_(f) (mg) Load F_(f) (g) F_(f) (g) Fiber 11.392 0.294 Fiber 1 0.065 0.070 Fiber 2 1.126 0.213 Fiber 2 0.043 0.051Fiber 3 0.937 0.486 Fiber 3 0.109 0.041 Fiber 4 1.644 0.221 Fiber 40.108 0.057 Mean 1.275 0.304 0.081 0.055

The peptide-based conditioner had a lower average friction than the DowCorning® 929 Cationic Emulsion conditioner in both cases. Subsequently,a conditioning sample of 1.5% lecithin was tested for fiber friction(low load) and the average mean frictional force was 3.366 mg,indicating that the conditioning effects observed with the peptide-basedconditioner was not due to the presence of the lecithin in theformulation. These results demonstrate the effectiveness of thepeptide-based hair conditioner.

Example 18 Preparation of a Peptide-Based Hair Colorant

The purpose of this Example was to prepare a peptide-based hair colorantby covalently attaching the D21 hair-binding peptide (SEQ ID NO:46) toDisperse Orange 3 dye. The dye was first functionalized with isocyanateand then reacted with the D21 peptide.

Functionalization of Disperse Orange 3:

In a dry box, 14.25 g of Disperse Orange 3 (Aldrich) was suspended in400 mL of dry THF in an addition funnel. A 2-liter, four-neck reactionflask (Corning Inc., Corning, N.Y.; part no. 1533-12), containing amagnetic stir bar, was charged with 200 mL of dry toluene. The flask wasfitted with a cold finger condenser (Corning Inc., part no. 1209-04) andwith a second cold finger condenser with an addition funnel, and wasplaced on an oil bath in a hood.

Phosgene (25.4 mL) was condensed into the reaction flask at roomtemperature. After phosgene addition was complete, the temperature ofthe oil bath was raised to 80° C. and the Disperse Orange 3 suspensionwas added to the reaction flask dropwise in 100 mL increments over 2 h,while monitoring the reaction temperature and gas discharge from thescrubber. The temperature was maintained at or below 64° C. throughoutthe addition. After addition was complete, the reactants were heated at64° C. for 1 h and then allowed to cool to room temperature withstirring overnight.

The reaction solvents were vacuum-distilled to dryness, whilemaintaining the contents at or below 40° C., and vacuum was maintainedfor an additional hour. The reaction flask was transferred to a dry box;the product was collected and dried overnight (15.65 g). The desiredproduct was confirmed by proton NMR.

Coupling of Isocyanate Functionalized Dye with D21 Hair-Binding Peptide:

Isocyanate functionalized Disperse Orange 3[(2-(4-isocyanatophenyl)-1-(4-nitrophenyl)diazene] (16 mg), prepared asdescribed above, was dissolved in 5 mL of DMF and added to a solutioncontaining 75 mg of non-protected D21 peptide (SEQ ID NO:46), obtainedfrom SynPep, dissolved in 10 mL of DMF. The solution was stirred at roomtemperature for 24 h. The solvent was evaporated yielding 91 mg of apurplish powder. The product was analyzed by MALDI mass spectrometry andwas found to have a molecular weight of 1766 g/mol, consistent withcovalent attachment of the dye molecule to the peptide.

Example 19 Selection of Tooth-Binding Peptides Using Biopanning

The purpose of this prophetic Example is to describe how to identifyphage peptides that bind to teeth with high affinity.

Extracted human teeth, which may be obtained from a Dental Office, arecleaned by brushing with soap solution, rinsed with deionized water, andallowed to air-dry at room temperature. The teeth are placed in a 15 mLcentrifuge tube (Corning Inc., Acton, Mass.), one tooth per tube. Theteeth samples are treated for 1 h with blocking buffer consisting of 1mg/mL BSA in TBST-0.5%, and then washed with TBST-0.5%. The teethsamples are incubated with the phage library (Ph.D-12 Phage DisplayPeptide Library Kit) and washed 10 times using the same conditionsdescribed in Example 1. After the acidic elution step, described inExample 1, the teeth samples are washed three more times with theelution buffer and then washed three times with TBST-0.5%. Theacid-treated teeth, which have acid resistant phage peptides stillattached, are used to directly infect E. coli ER2738 cells as describedin Example 2. The amplified and isolated phages are contacted with afresh tooth sample and the biopanning procedure is repeated two moretimes. After the third round of biopanning, the acid-treated teeth areused to directly infect E. coli ER2738 cells, and the cells are culturedas described in Example 1. Single black plaques are randomly picked forDNA isolation and sequence analysis. The single plaque lysates areprepared following the manufacturer's instructions (New England Biolabs)and the single stranded phage genomic DNA is purified using the QIAprepSpin M13 Kit (Qiagen, Valencia, Calif.) and sequenced using −96 gIIIsequencing primer, as described in Example 1.

The identified peptide sequences will have a binding affinity for teeth.The binding specificity and affinity of the identified tooth-bindingpeptides is determined as described in Example 6.

Example 20 Preparation of a Peptide-Based Tooth Whitener

The purpose of this prophetic Example is to describe how to prepare apeptide-based tooth whitener by coupling a tooth-binding peptide to thewhite pigment, titanium dioxide.

Dry titanium dioxide having an average particle size less than 2 μm(available from E.I. du Pont de Nemours and Co., Wilmington, Del.) istreated with a solution of 3-aminopropyltriethoxysilane (available fromAldrich) in dry acetone to covalently attach amino groups to the surfaceof the titanium dioxide. The excess reagent is removed by decantationafter centrifuging to settle out the particles. The resultant particlesare then treated with sufficient glutaraldehyde (available from SigmaChemical Co.) to react with the surface attached amino groups.

A tooth binding peptide, identified using the method described inExample 19, is obtained from SynPep. The tooth-binding peptide sequenceis terminated with 1-5 lysine residues at the C terminus. Thetooth-binding peptide is then added to the glutaraldehyde-treatedtitanium dioxide particles and is covalently coupled to the pendant freealdehyde groups on glutaraldehyde through an amine group on the peptide.

Example 21 Selection of Tooth Pellicle Binding Peptides Using StandardBiopanning

The purpose of this Example was to identify phage peptides that bindtooth pellicle using standard phage display biopanning.

The compressed HAP disks (Hydroxy Apatite disk, 3 mm diameter) were usedto form the pellicles by incubating the disks inside a human mouth for1.5 hours followed by TBS rinse. The disks were then incubated inSUPERBLOCK® blocking buffer (Pierce Chemical Company, Rockford, Ill.;Prod. #37535) for 1 hour at room temperature, followed by 3 washes withTBST (TBS in 0.05% TWEEN® 20). Libraries of phage containing randompeptide inserts (10¹¹ pfu) from 7 to 20 amino acids were added to eachtube. After 60 minutes of incubation at room temperature and shaking at50 rpm, unbound phage were removed by aspirating the liquid out of eachwell followed by 6 washes with 1.0 mL TBS containing the detergentTWEEN® 20 (TBST) at a final concentration of 0.05%.

The sample disks were then transferred to clean tube and 200 μL ofelution buffer consisting of 1 mg/mL BSA in 0.2 M glycine-HCl, pH 2.2,was added to each well and incubated for 10 min to elute the boundphages. Then, 32 μL of neutralization buffer consisting of 1 M Tris-HCl,pH 9.2, was added to each well. The phage particles, which were in theelution buffer as well as on the sample disks, were amplified byincubating with diluted E. coli ER2738 cells, from an overnight culturediluted 1:100 in LB medium, at 37° C. for 4.5 h. After this time, thecell culture was centrifuged for 30 s and the upper 80% of thesupernatant was transferred to a fresh tube, ⅙ volume of PEG/NaCl (20%polyethylene glyco-800, 2.5 M sodium chloride) was added, and the phagewas allowed to precipitate overnight at 4° C. The precipitate wascollected by centrifugation at 10,000×g at 4° C. and the resultingpellet was resuspended in 1 mL of TBS. This was the first round ofamplified stock. The amplified first round phage stock was then titeredaccording to the standard protocol. For the 2^(nd), 3^(rd) and 4^(th)round of biopanning, more than 2×10¹¹ pfu of phage stock from theprevious round was used. The biopanning process was repeated for 2 morerounds under the same conditions as described above. The same biopanningcondition was used for the 4^(th) round, except the washing solution wasTBS with 0.5% Tween20 instead of 0.05% Tween20.

After the 4^(th) round of biopanning, 95 random single phage plaquelysates were prepared following the manufacture's instructions (NewEngland Biolabs) and the single stranded phage genomic DNA was purifiedusing the QIAprep Spin M13 Kit (Qiagen, Valencia, Calif.) and sequencedat the DuPont Sequencing Facility using −96 gIII sequencing primer(5′-CCCTCATAGTTAGCGTAACG-3′), given as SEQ ID NO: 105. The displayedpeptide is located immediately after the signal peptide of gene III.Based on the peptide sequences, 20 phage candidates were selected forfurther pellicle binding analysis.

Example 22 Characterization of Tooth Pellicle Binding Candidates onPellicle Surface

Total of 20 selected phage candidates were used in phage ELISA

Experiment. Purified phage lysates were used for binding to pellicle HAPdisks using an anti-M13 phage antibody conjugated tohorseradish-peroxidase, followed by the addition of chromogenic agentTMB, obtained from Pierce Biotechnology (Rockford, Ill.). The plateswere read at A₄₅₀ nm.

For each phage candidate to be tested, the pellicle coated HAP disks (3mm diameter) was incubated for 1 h at room temperature with 200 μL ofblocking buffer, consisting of 2% non-fat dry milk (Schleicher &Schuell, Inc.) in TBS. The blocking buffer was removed by aspirating theliquid out of each tube. The tube was rinsed 6 times with wash bufferconsisting of TBST-0.05%. The wells were filled with 200 μL of TBST-0.5%containing 1 mg/mL BSA and then 10 μL (over 10¹⁰ pfu) of purified phagestock was added. The samples were incubated at room temperature for 60min with slow shaking. The non-binding phage was removed by washing 6times with TBST-0.5%. Then, 100 μL of horseradish peroxidase/anti-M13antibody conjugate (Amersham USA, Piscataway, N.J.), diluted 1:500 inthe blocking buffer, was added and incubated for 1 h at roomtemperature. The conjugate solution was removed and was washed 6 timeswith TBST-0.5%. TMB substrate (200 μL), obtained from PierceBiotechnology (Rockford, Ill.) was added to each well and the color wasallowed to develop for between 5 to 30 min, typically for 10 min, atroom temperature. Then, stop solution (200 μL of 2 M H₂SO₄) was added toeach well and the solution was transferred to a 96-well plate and theA₄₅₀ was measured using a microplate spectrophotometer (MolecularDevices, Sunnyvale, Calif.). The resulting absorbance values,) are givenin Table 16.

TABLE 16 Amino Acid O.D. at SEQ ID ID Sequences 450 nm NO: ControlNo phage 0.218 Pell 1 AHPESLGIKYALDGNSDPHA 0.739 106 Pell 2ASVSNYPPIHHLATSNTTVN 0.75 107 Pell 3 DECMEPLNAAHCWR 0.49 108 Pell 4DECMHGSDVEFCTS 0.664 109 Pell 5 DLCSMQMMNTGCHY 0.83 110 Pell 6DLCSSPSTWGSCIR 0.735 111 Pell 7 DPNESNYENATTVSQPTRHL 0.831 112 Pell 8EPTHPTMRAQMHQSLRSSSP 0.712 113 Pell 9 GNTDTTPPNAVMEPTVQHKW 0.755 114Pell 10 NGPDMVQSVGKHKNS 0.729 115 Pell 11 NGPEVRQIPANFEKL 0.607 116Pell 12 NNTSADNPPETDSKHHLSMS 0.521 117 Pell 13 NNTWPEGAGHTMPSTNIRQA0.598 118 Pell 14 NPTATPHMKDPMHSNAHSSA 0.7 119 Pell 15NPTDHIPANSTNSRVSKGNT 0.567 120 Pell 16 NPTDSTHMMHARNHE 0.578 121 Pell 17QHCITERLHPPCTK 0.614 122 Pell 18 TPCAPASFNPHCSR 0.416 123 Pell 19TPCATYPHFSGCRA 0.731 124 Pell 20 WCTDFCTRSTPTSTSRSTTS 0.715 125

Example 23 Selection of Tooth Enamel Binding Peptides Using StandardBiopanning

The purpose of this example was to identify phage peptides that bindtooth enamel using standard phage display biopanning.

The unpolished bovine enamel blocks from incisors (3 mm squares) andpolished bovine enamel disks from the incisors (3 mm diameter disks)were embedded in wax for form a well with only the intended surfaceswere exposed. The enamel surfaces were then incubated in SUPERBLOCK®blocking buffer (Pierce Chemical Company, Rockford, Ill.; Prod. #37535)for 1 hour at room temperature, followed by 3 washes with TBST (TBS in0.05% TWEEN® 20). Libraries of phage containing random peptide inserts(10¹¹ pfu) from 7 to 20 amino acids were added to the enamel well for 10minutes pre-absorption to titrate the wax surface, unbound phage wereremoved by aspirating the liquid out of each well. Then, 100 μL of thesame phage library (10¹¹ pfu) was added to the enamel well for 60 minincubation at room temperature with slow 50 rpm shaking, followed by 6washes with 1.0 mL TBS containing the detergent TWEEN® 20 (TBST) at afinal concentration of 0.05%.

The enamel blocks (or polished disks) were then cut out of the wax welland transferred to a clean tube and 200 μL of elution buffer consistingof 1 mg/mL BSA in 0.2 M glycine-HCl, pH 2.2, was added to each well andincubated for 10 min to elute the bound phages. Then, 32 μL ofneutralization buffer consisting of 1 M Tris-HCl, pH 9.2, was added toeach tube. The phage particles, which were in the elution buffer as wellas on the enamel blocks, were amplified by incubating with diluted E.coli ER2738 cells, from an overnight culture diluted 1:100 in LB medium,at 37° C. for 4.5 h. After this time, the cell culture was centrifugedfor 30 s and the upper 80% of the supernatant was transferred to a freshtube, ⅙ volume of PEG/NaCl (20% polyethylene glyco-800, 2.5 M sodiumchloride) was added, and the phage was allowed to precipitate overnightat 4° C. The precipitate was collected by centrifugation at 10,000×g at4° C. and the resulting pellet was resuspended in 1 mL of TBS. This wasthe first round of amplified stock. The amplified first round phagestock was then titered according to the standard protocol. For the2^(nd) round of biopanning, more than 2×10¹¹ pfu of phage stock from theprevious round was used. The biopanning process was repeated for 1 morerounds under the same conditions as described above. The same biopanningcondition was used for the 3^(rd) round, except the washing solution wasTBS with 0.5% Tween20 instead of 0.05% Tween20.

After the 3^(rd) round of biopanning, 95 random single phage plaquelysates were prepared following the manufacture's instructions (NewEngland Biolabs) and the single stranded phage genomic DNA was purifiedusing the QIAprep Spin M13 Kit (Qiagen, Valencia, Calif.) and sequencedat the DuPont Sequencing Facility using −96 gIII sequencing primer(5′-CCCTCATAGTTAGCGTAACG-3′), given as SEQ ID NO: 105. The displayedpeptide is located immediately after the signal peptide of gene III.Based on the peptide sequences, 20 phage candidates were selected forfurther pellicle binding analysis (Table 17). BoEn means bovine enameland BoEn P means polished bovine enamel.

TABLE 17 Bovine Enamel Binding Peptide Sequences ID Amino Acid SequenceSEQ ID NO BoEn P1 APPLKTYMQERELTMSQNKD 126 BoEn P2 EPPTRTRVNNHTVTVQAQQH127 BoEn P3 GYCLRGDEPAVCSG 128 BoEn P4 LSSKDFGVTNTDQRTYDYTT 129 BoEn P5NFCETQLDLSVCTV 130 BoEn P6 NTCQPTKNATPCSA 131 BoEn P7PSEPERRDRNIAANAGRFNT 132 BoEn P8 THNMSHFPPSGHPKRTAT 133 BoEn P9TTCPTMGTYHVCWL 134 BoEn P10 YCADHTPDPANPNKICGYSH 135 BoEn 1AANPHTEWDRDAFQLAMPPK 136 BoEn 2 DLHPMDPSNKRPDNPSDLHT 137 BoEn 3ESCVSNALMNQCIY 138 BoEn 4 HNKADSWDPDLPPHAGMSLG 139 BoEn 5LNDQRKPGPPTMPTHSPAVG 140 BoEn 6 NTCATSPNSYTCSN 141 BoEn 7 SDCTAGLVPPLCAT142 BoEn 8 TIESSQHSRTHQQNYGSTKT 143 BoEn 9 VGTMKQHPTTTQPPRVSATN 144BoEn 10 YSETPNDQKPNPHYKVSGTK 145

Example 24 Characterization of Tooth Enamel Binding Peptide Candidateson Enamel Surface

Total of 20 selected phage candidates were used in phage ELISAExperiment. Purified phage lysates were used for binding to the enamelblocks using an anti-M13 phage antibody conjugated tohorseradish-peroxidase, followed by the addition of chromogenic agentTMB, obtained from Pierce Biotechnology (Rockford, Ill.). The plateswere read at A₄₅₀ nm.

For each phage candidate to be tested, the polished and unpolishedenamel blocks were incubated for 1 h at room temperature with 200 μL ofblocking buffer, consisting of 2% non-fat dry milk (Schleicher &Schuell, Inc.) in TBS. The blocking buffer was removed by aspirating theliquid out of each tube. The tube was rinsed 6 times with wash bufferconsisting of TBST-0.05%. The wells were filled with 200 μL of TBST-0.5%containing 1 mg/mL BSA and then 10 μL (over 10¹⁰ pfu) of purified phagestock was added. The samples were incubated at room temperature for 60min with slow shaking. The non-binding phage was removed by washing 6times with TBST-0.5%. Then, 100 μL of horseradish peroxidase/anti-M13antibody conjugate (Amersham USA, Piscataway, N.J.), diluted 1:500 inthe blocking buffer, was added and incubated for 1 h at roomtemperature. The conjugate solution was removed and was washed 6 timeswith TBST-0.5%. TMB substrate (200 μL), obtained from PierceBiotechnology (Rockford, Ill.) was added to each well and the color wasallowed to develop for between 5 to 30 min, typically for 10 min, atroom temperature. Then, stop solution (200 μL of 2 M H₂SO₄) was added toeach well and the solution was transferred to a 96-well plate and theA₄₅₀ was measured using a microplate spectrophotometer (MolecularDevices, Sunnyvale, Calif.). The resulting absorbance values,) are givenin Table 18 and 19. BoEn means bovine enamel and BoEn P means polishedbovine enamel.

TABLE 18 Phage ELISA Results on Bovine EnamelBinding Assay of Selected Phage Candidates O.D. at SEQ ID IDAmino Acid Sequence 450 nm NO: Control no phage 0.112 BoEn P2EPPTRTRVNNHTVTVQAQQH 0.641 127 BoEn P3 GYCLRGDEPAVCSG 0.665 128 BoEn P5NFCETQLDLSVCTV 0.797 130 BoEn P6 NTCQPTKNATPCSA 0.83 131 BoEn P8THNMSHFPPSGHPKRTAT 2.02 133

TABLE 19 Phage ELISA Results on Bovine Polished EnamelBinding Assay of Selected Phage Candidates O.D. at SEQ ID IDAmino Acid Sequence 450 nm NO: Control no phage 0.193 BoEn 1AANPHTEWDRDAFQLAMPPK 1.402 136 BoEn 5 LNDQRKPGPPTMPTHSPAVG 0.944 140BoEn 6 NTCATSPNSYTCSN 2.38 141 BoEn 7 SDCTAGLVPPLCAT 0.892 142 BoEn 9VGTMKQHPTTTQPPRVSATN 0.568 144 BoEn 10 YSETPNDQKPNPHYKVSGTK 3.942 145

1. (canceled)
 2. A tooth enamel-binding peptide having an amino acidsequence selected from the group consisting of SEQ ID NOs:126-145.
 3. Adiblock, peptide-based oral care reagent having the general structure(OBP)_(n)-OBA, wherein a) OBP is an oral cavity surface-binding peptide;b) OBA is an oral care benefit agent; and c) n ranges from 1 to about10,000 wherein said oral cavity surface-binding peptide is atooth-binding peptide having affinity for tooth enamel; wherein saidtooth-binding peptide is from about 7 to about 25 amino acids and has abinding affinity, measured as MB₅₀, equal to or less than 10⁻⁵ M.
 4. Atriblock, peptide-based oral care reagent having the general structure[(OBP)_(m)-S]_(n)-OBA, wherein a) OBP is an oral cavity surface-bindingpeptide; b) OBA is an oral care benefit agent; c) S is a spacer; d) mranges from 1 to about 50; and e) n ranges from 1 to about 10,000wherein said oral cavity surface-binding peptide is a tooth-bindingpeptide having affinity for tooth enamel; wherein said tooth-bindingpeptide is from about 7 to about 25 amino acids and has a bindingaffinity, measured as MB₅₀, equal to or less than 10⁻⁵ M. 5-9.(canceled)
 10. The peptide-based oral care reagent according to eitherclaim 3 or claim 4 wherein the oral cavity surface-binding peptidecomprises a cysteine residue on at least one end of the peptidesequence.
 11. The peptide-based oral care reagent of claim 4 wherein thespacer is selected from the group consisting of ethanol amine, ethyleneglycol, polyethylene with a chain length of 6 carbon atoms, polyethyleneglycol with 3 to 6 repeating units, phenoxyethanol, propanolamide,butylene glycol, butyleneglycolamide, propyl phenyl chains, ethyl alkylchains, propyl alkyl chains, hexyl alkyl chains, steryl alkyl chains,cetyl alkyl chains, and palmitoyl alkyl chains.
 12. The peptide-basedoral care reagent of claim 4 wherein the spacer is a peptide comprisingamino acids selected from the group consisting of glycine, alanine,serine, and mixtures thereof.
 13. The peptide-based oral care reagentaccording to claim 3 or claim 4 wherein the benefit agent is selectedfrom the group consisting of white colorants, whitening agents, enzymes,anti-plaque agents, anti-staining agents, anti-microbial agents,anti-caries agents, flavoring agents, coolants, and salivating agents.14. The peptide-based oral care reagent according to claim 13 whereinthe white colorants or whitening agent is selected from the groupconsisting of hydroxyapatite, zirconium silicate, titanium dioxide, andtitanium dioxide nanoparticles.
 15. The peptide-based oral care reagentaccording to claim 13, wherein the enzyme is selected from the groupconsisting of oxidases, peroxidases, proteases, lipases, glycosidases,esterases, and polysaccharide hydrolases.
 16. The peptide-based oralcare reagent according to claim 13 wherein anti-plaque agents areselected from the group consisting of fluoride ion sources andanti-microbial agents.
 17. The peptide-based oral care reagent accordingto claim 13 wherein the flavoring agents are selected from the groupconsisting of oil of wintergreen, oil of peppermint, oil of spearmint,menthol, methyl salicylate, eucalyptol, and vanillin.
 18. Thepeptide-based oral care reagent of claim 4 wherein the spacer is apeptide comprising the amino acid sequence as set forth in SEQ ID NO:65.19. An oral care reagent composition comprising an effective amount ofthe peptide-based oral care reagent of claim 3 or claim
 4. 20. The oralcare reagent composition according to claim 19 selected from the groupconsisting of toothpaste, dental cream, gel or tooth powder, mouth wash,breath freshener, and dental floss.
 21. The oral care reagentcomposition according to claim 20 wherein the composition optionallycomprises a reagent selected from the group consisting of abrasives,surfactants, chelating agents, fluoride sources, thickening agents,buffering agents, solvents, humectants, carriers, and bulking agents.22. A method for applying a oral care benefit agent to an oral surfacecomprising contacting the oral surface with the peptide-based oral carereagent of either of claim 3 or 4 under conditions whereby thepeptide-based oral care reagent adheres to the oral surface. 23.(canceled)